Report on a Validation Study of the IL-2 Luc leukocyte toxicity test (IL-2 Luc
LTT)

MITA Validation Management Team1

1. Summary....................................................................................................................... 6
3. Background................................................................................................................. 13
3-1. What is immunotoxicity? ................................................................................................. 13
3-2. The current status of in vitro immunotoxicity tests .......................................................... 14
3-3. Predictivity of in vitro immunotoxicity tests.................................................................... 15
3-4. Multi-ImmunoTox Assay (MITA) .................................................................................... 16
3-5. The evaluation of immunotoxicity profiles of well-known immunosuppressive drugs by
the MITA ................................................................................................................................. 18
3-7. The IL-2 Luc assay plays a principal role in detecting the immunosuppressive effects ofchemicals in the MITA ............................................................................................................ 20
3-8. The IL-2 Luc assay cannot detect immunosuppressive effects of drugs which aredependent on the inhibition of DNA synthesis or anti-proliferative effects ............................ 20
3-9. The IL-2 Luc LTT can detect antimitotic effects of chemicals. ....................................... 21
3-10. The process of validation of the combined assays of the IL-2 Luc assay and the IL-2
Luc LTT ............................................................................................................................... 23

4. Test method and modification .................................................................................... 24
4-1. IL-2 reporter cell line 2H4 ............................................................................................... 24
4-2. Chemical treatment of 2H4 cells and measurement of luciferase activity ....................... 24
4-3. Criteria to determine antimitotic effects of chemicals ..................................................... 26
4-4. Bioluminescence system .................................................................................................. 28

5. Validation Management Structure .............................................................................. 31
5-1. Validation Management Team (VMT) ............................................................................. 31
5-2. Management office........................................................................................................... 32
5-3. Meetings held ................................................................................................................... 32

6. Study Design .............................................................................................................. 33
7. Test Chemicals ............................................................................................................ 34
7-1. Basic rules for chemical selection .................................................................................... 34
7-1-1. Applied selection criteria .......................................................................................... 35
7-1-2. Chemical acquisition, coding and distribution .......................................................... 36
7-1-3. Handling.................................................................................................................... 36
7-2. Pre-validation - Phase 0 trial ......................................................................................... 37
7-3. Validation study - Phase I trial ......................................................................................... 37
7-4. Validation study - Phase II trial ........................................................................................ 382

8. Protocols ..................................................................................................................... 40
8-1. Overview of IL-2 Luc LTT .............................................................................................. 40
8-2. Protocol for IL-2 Luc LTT ............................................................................................... 41
8-2-1. Reagents and equipment ........................................................................................... 41
8-2-2. Culture media ............................................................................................................ 43
8-2-3. Preparation of stimulant for 2H4 cells ...................................................................... 44
8-2-4. Thawing of 2H4 cells ................................................................................................ 45
8-2-5. Maintenance of 2H4 cells ......................................................................................... 45
8-2-6. Preparation of cells for assay .................................................................................... 46
8-2-7. Preparation of chemicals and cell treatment with chemicals .................................... 46
8-2-8. Dilution of chemicals ................................................................................................ 48
8-2-9. Measurements ........................................................................................................... 48
8-2-10. Luminometer apparatus .......................................................................................... 49
8-2-11. Positive and negative controls ................................................................................. 49
8-2-12. Calculation and definition of parameters for IL-2 Luc LTT ................................... 49
8-2-13. Acceptance criteria .................................................................................................. 50
8-2-14. Criteria .................................................................................................................... 51
8-3. Data collection ................................................................................................................. 52
8-3-1. Operating procedure.................................................................................................. 52
8-3-2. Chemicals.................................................................................................................. 53
8-3-3. Data handling ............................................................................................................ 53
8-3-4. Index from each experiment and decision criteria for judgment .............................. 54
8-3-5. Reliability.................................................................................................................. 56
8-3-6. Predictivity ................................................................................................................ 57
8-4. Quality assurance ............................................................................................................. 58

9. Results ........................................................................................................................ 58
9-1. Final criteria for Phase I trial ........................................................................................... 59
9-1-1. Acceptance criteria .................................................................................................... 59
9-1-2. Criteria ...................................................................................................................... 60
9-1-3. Predictivity ................................................................................................................ 61
9-2. Phase 0 trial (for technical transfer) ................................................................................. 63
9-3. Phase I trial (for within- and between-laboratory reproducibility) .................................. 63
9-3-1. Test conditions .......................................................................................................... 633

9-3-2. Within-laboratory variation assessments in Phase I trial .......................................... 64
9-3-3. Between-laboratory variation assessments in Phase I trial ....................................... 64
9-4. Phase II trial (for between-laboratory reproducibility and predictivity) .......................... 65
9-4-1. Test conditions .......................................................................................................... 65
9-4-2. Between-laboratory variation assessments in Phase II trial ...................................... 66
9-5. Quality assurance (Appendix 16) ..................................................................................... 67
9-6. Combined results of Phase I and II trials (for between- and within- laboratory
reproducibility) ........................................................................................................................ 69
9-6-1. Test conditions .......................................................................................................... 69
9-6-2. Between-laboratory variation assessments from Phase I and II trials (Table 17) ..... 69
9-7. The predictivity of the IL-2 Luc LTT in the Phase I and Phase II trials .......................... 72

10. Discussion................................................................................................................. 75
10-1. Reliability ....................................................................................................................... 75
10-2. Between- and within-laboratory reproducibility ............................................................ 76
10-3. Predictivity ..................................................................................................................... 76
10-3-1. The predictivity of the IL-2 Luc LTT in the validation trials .................................. 76
10-3-2. IL-2 Luc LTT judgment for 83 data set chemicals.................................................. 76
10-3-3. Application of the IL-2 Luc LTT with IL-2 Luc as part of an IATA approach ....... 78
10-4. The possible reason for the false negative judgment for carmustin, paclitaxel,
leflunomide, and methotrexate (MTX) by the IL-2 Luc LTT. ................................................. 87
10-5. Limitation and applicability domain of the IL-2 Luc LTT ............................................. 88
10-6. The possible reason for the poor specificity of the IL-2 Luc LTT ................................. 89
10-7. Regulatory application of the IL-2 Luc LTT .................................................................. 92

11. Conclusion ................................................................................................................ 93
12. Acknowledgement .................................................................................................... 94
13. References ................................................................................................................ 95
14. List of abbreviations ................................................................................................. 98

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List of Appendixes.
Appendix 01 IL-2 Luc LTT Study plan Ver1.0
Appendix 02-1 Coded chemicals
Appendix 02-2 Coded chemicals
Appendix 03 Chemical structure of the test chemicals for the Phase 0 trial_IL-2 Luc
LTT
Appendix 04 Chemical structure of the test chemicals for the Phase I trial_IL-2 Luc
LTT
Appendix 05 Chemical structure of the test chemicals for the Phase II trial_IL-2 Luc
LTT
Appendix 06 IL-2 Luc LTT protocol Ver.001.4
Appendix 07 Principle of measurement of luciferase activity
Appendix 08 Validation of reagents and equipment
Appendix 09 Data sheet for MITA Il-2 Luc LTT Ver.008.8 20210112
Appendix 10 MITA IL-2 Luc LTT Phase I results trial
Appendix 11 MITA IL-2 Luc LTT Phase II results trial
Appendix 12 Supplementary Table 1 IL-2 Luc LTT
Appendix 13 Reference 25 chemicals_ text
Appendix 14 Supplementary Table 2 IL-2 Luc LTT
Appendix 15 Reference 63 chemicals_ text
Appendix 16 QC for IL-2 Luc LTT validation (R1)
Appendix 17-1 IL-2 Luc LTT Check List
Appendix 17-2 IL-Luc LTT Check List (1)51. Summary
The IL-2 Luciferase leukocyte toxicity test (IL-2 Luc LTT) was developed using
one of the luciferase reporter assays that comprise the Multi-ImmunoTox Assay(MITA), a high-throughput screening system that our group developed to evaluate
chemical immunotoxicity. The MITA assesses effects of chemicals on promoter activityof several cytokines (i.e., IL-1, IL-2, IFN-γ, and IL-8). Although the final long-termgoal is to officially validate the MITA for within- and between-laboratory
reproducibility and predictivity, so far the validation studies for the individual IL-2
luciferase assay (IL-2 Luc assay), the IL-1 luciferase assay (IL-1 Luc assay), and, asreported here, the IL-2 Luc LTT have been conducted step by step. This report
describes the results of the validation study for the IL-2 Luc LTT.
In the MITA, we used three stable reporter cell lines transfected with luciferase
genes under the control of IL-2 and IFN-β, IL-8, or IL-1βpromoters. The IL-2 Luc
LTT used 2H4 cells from among these cell lines, which were derived from Jurkat cells
containing stable luciferase green (SLG) regulated by the IL-2 promoter, stableluciferase orange (SLO) regulated by the IFN-β promoter, and stable luciferase red(SLR) regulated by the glycerol-3-phosphate dehydrogenase (GAPDH) promoter.
The lead laboratory reported that the IL-2 Luc assay using 2H4 cells can detect theeffect of chemicals on IL-2 transcription and hence the immunotoxicity of chemicals,
mainly by affecting T cell function. In addition, the validation study for the IL-2 Luc
assay conducted by three independent laboratories showed reasonable performance of6the assay. However, it was found that immunotoxicity due to antimitotic effects of thechemicals could not be detected by the IL-2 Luc assay.
To overcome this drawback of the IL-2 Luc assay, the lead laboratory attempted toestablish a new assay to detect antimitotic effects of chemicals by taking advantage ofthe triple reporter cell properties of 2H4 cells. To begin with, it was hypothesized that if
2H4 cells are treated with antimitotic agents for 24 h and then stimulated with PMA(phorbol myristate acetate) /Io (ionomycin), they might reduce GAPDH promoterdriven luciferase activity (GAPLA) without suppressing IL-2 promoter-driven
luciferase activity (IL2LA). We first determined the optimal incubation time with drugs
and the seeding cell density, and then, the criteria to detect antimitotic effects ofchemicals, namely the reduction of GAPLA without reducing IL2LA. We designed theassay as the IL-2 luciferase leukocyte toxicity test (IL-2 Luc LTT).
In the validation study of the IL-2 Luc LTT, the preliminary test trial (Phase 0) was
performed by the participating laboratories following explicit explanations of “IL-2 Luc
leukocyte toxicity test (IL-2 Luc LTT) protocol ver.001.2” by the lead laboratory,
Tohoku University. Three laboratories participated in the Phase 0 trial of the IL-2 Luc
LTT using three open-labeled chemicals, namely bleomycin sulfate, dexamethasone,and 6-thioguanine, and conducted one set of three experiments for each chemical. The
response patterns for the three chemicals were similar among the three laboratories.
Based on these results, the validation management team (VMT) judged the technicaland protocol transfer of the IL-2 Luc LTT as acceptable.

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In the Phase I trial, a total of five coded chemicals were evaluated by threeexperimental sets based on the IL-2 Luc LTT protocol ver.001.3. The within-laboratory
reproducibility was 100.0% (15/15). The between-laboratory reproducibility was
100.0% (5/5).
In the Phase II trial, a total of 20 coded chemicals were evaluated by one experiment
set based on the IL-2 Luc LTT protocol ver.001.4. The between-laboratory
reproducibility was 90.0 % (18/20).
In the combined results of the Phase I and II trials, the average within-laboratory
reproducibility was 100.0% (15/15). The between laboratory reproducibility was 92.0%(23/25).
The predictivity of the IL-2 Luc LTT was determined for detecting antimitotic
effects and for immunosuppression by chemicals. To determine the predictivity of theassay, the lead laboratory first surveyed the literature, generated reference data for
immunotoxic profiles of chemicals used in the validation study and the data set. On theother hand, to determine whether chemicals have antimitotic effects or
immunosuppression, the lead laboratory made a list of criteria based on the rationale for
immunotoxic classification of chemicals proposed by Luster et al. (Luster et al., 1992).
Briefly, Luster et al. (1992) presented a panel of immunotoxicity tests and demonstrated
that the combination of 3 or more different immunotoxicity tests resulted in 100 %concordance for the judgment of immunotoxicity. The list of criteria we used was basedon immunotoxic information and immunotoxicity parameters presented by Luster et al.
(1992).
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When the performance of the IL-2 Luc LTT was determined with reference to theclassification of chemicals with antimitotic effects, it was 76.5 % (13/17) for meansensitivity, 75.0 % (6/8) for mean specificity, and 76.0 % (19/25) for mean predictivityof the combined data from the Phase I and Phase II trials. When the performance of the
IL-2 Luc LTT was determined with reference to the classification of chemicals withimmunosuppressive effects, it was 71.4 % (15/21) for mean sensitivity, 100.0 % (4/4)
for mean specificity, and 76.0 % (19/25) for mean prediction of the combined data fromthe Phase I and Phase II trials.
To further characterize the IL-2 Luc LTT assay, the lead laboratory assessed a totalof 83 test compounds including 46 pharmaceutical drugs that were composed of 12 anticancer drugs, 8 immunosuppressive drugs with antimitotic effects, 8
immunosuppressive drugs without antimitotic effects, 3 JAK inhibitors, and 15 nonimmunosuppressive drugs; and 37 non-pharmaceutical chemicals (23immunosuppressive and 14 non-immunosuppressive). Seven of the 23
immunosuppressive chemicals have antimitotic properties.
When pharmaceutical drugs and non-pharmaceutical chemicals were examined
together, the performance of the IL-2 Luc LTT for antimitotic effects was 63.0 %(17/27) for sensitivity, 90.2 % (46/51) for specificity, and 80.8 % (63/78) for
predictivity. When the applicability domain was taken into consideration, theperformance of the IL-2 Luc LTT for antimitotic effects was 66.7 % (16/24) for
sensitivity, 90.2 % (46/51) for specificity, and 79.5 % (62/78) for predictivity.

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We next examined whether the combination of the IL-2 Luc LTT and the IL-2 Luc
assay improves the performance of the IL-2 Luc LTT for immunosuppressive effects aspart of an integrated approaches to testing and assessment (IATA) approach. Whenpharmaceutical drugs were examined, the performance of the IL-2 Luc assay alone for
immunosuppressive effects was 61.3 % (19/31) for sensitivity, 53.3 % (8/15) for
specificity, and 58.7 % (27/46) for predictivity. When non-pharmaceutical chemicals
were examined, the performance of the IL-2 Luc assay alone for immunosuppressive
effects was 65.2 % (15/23) for sensitivity, 64.3 % (9/14) for specificity, and 64.9 %(24/37) for predictivity. When pharmaceutical drugs and non-pharmaceutical chemicals
were examined together, the performance of the IL-2 Luc assay alone for
immunosuppressive effects was 63.0 % (34/54) for sensitivity, 58.6 % (17/29) for
specificity, and 61.4 % (51/83) for predictivity.
When pharmaceutical drugs were examined, the performance of the combined IL-2
Luc + IL-2 Luc LTT assay for immunosuppressive effects was 93.3 % (28/30) for
sensitivity, 50.0 % (7/14) for specificity, and 79.5 % (35/44) for predictivity. When
non-pharmaceutical chemicals were examined, the performance of the combined IL-2
Luc + IL-2 Luc LTT assay for systemic immunosuppressive effects was 82.6 % (19/23)
for sensitivity, 58.3 % (7/12) for specificity, and 74.3 % (26/35) for predictivity. Whenpharmaceutical drugs and non-pharmaceutical chemicals were examined together, theperformance of the combined assay for systemic immunosuppressive effects was
88.7 % (47/53) for sensitivity, 53.8 % (14/26) for specificity, and 77.2 % (61/79) for
predictivity. After considering the applicability domain, the performance of the10combined IL-2 Luc + IL-2 Luc LTT assay was 90.0 % (45/50) for sensitivity, 53.8 %(14/26) for specificity, and 77.6% (59/76) for predictivity.
These results suggest that, although the IL-2 Luc LTT alone is not sufficient todetect the immunosuppressive effects of chemicals, the combination with the IL-2 Luc
assay can be a promising approach to detect immunosuppression by chemicals. Like theIL-2 Luc assay or the IL-8 Luc assay, chemicals that require metabolic activation or are
poorly water-soluble should be outside of the applicability domain. In this study, there
were 3 chemicals outside of the applicability domain. Even though these applicability
domains are taken into consideration, either the IL-2 Luc assay alone, or in combination
with the IL-2 Luc LTT cannot cover all the effects of chemicals on human immune
system. Therefore, it is imperative to further develop other in vitro systems to detect theeffects of chemicals on different aspects of immune response. By accumulating andcombining various approaches to detect chemical immunotoxicity, the in vitro assays
can cover the effects of chemicals on the broad range of human immune system.

2. Objective and rationale for the study
The objective of the present validation study was to determine the usefulness andlimitations of the IL-2 luciferase leukocyte toxicity test (IL-2 Luc LTT) as a non-animal
screening method to detect and assess the immunotoxicity of chemicals.
The specific objectives of the study were to establish:
1) “Transferability”, i.e., the extent to which a laboratory can adapt and easily
implement the IL-2 Luc LTT;112) “Between- or inter-laboratory reproducibility”, i.e., the extent to which results agree
among different laboratories;
3) "Within- or intra-laboratory reproducibility", i.e., the extent to which results agree inthe same laboratory; and
4) “Predictivity”, i.e., the extent to which the in vitro results agree with the known
immunotoxic profiles of the chemicals.
The rationale is multifaceted. The development of this assay is important because:
1) Environmental contaminants, food additives, and drugs can affect the immune
system, resulting in immune dysregulation;2) Immune dysregulation can have serious adverse health consequences, ranging fromreduced resistance to infection and neoplasia to allergic and autoimmune conditions;3) In vitro methods are needed to detect immune effects of chemicals due to the highcost, ethical concerns, and questionable relevance to risk assessment for humans
using animal cells and model systems;
4) An IATA approach needs to be developed since immune effects of chemicals areunlikely to be detected by a single in vitro assay;5) Antimitotic effects of chemicals could not be detected by the IL-2 Luc assay; and
6) The regulatory application is eventual generation of an OECD test method for
immunotoxicity.
IL-2 is a reasonable initial target to establish immunotoxicity testing as it is a
key cytokine in T cell activation, proliferation, and maintenance of various T cellsubsets. As noted, one of the first assays to be validated as part of the larger MITA
12

was the IL-2 Luc assay, which provided judgement of chemicals to be immunotoxic
through alteration (often suppression) of the IL-2 promoter. Surprisingly, many
chemicals that are known immunosuppressants (i.e., cyclophosphamide, azathioprine(AZ), mycophenolic acid (MPA), mizoribine (MZR), and methotrexate (MT)) had noeffect on IL-2 Luc. It was therefore hypothesized that chemicals that exhibit
immunosuppressive effects via antimitotic effects may instead be judged
immunosuppressive using the IL-2 Luc LTT assay. It is also proposed that the
combination of the IL-2 Luc with IL-2 Luc LTT can be part of an IATA approach toassess the immunotoxic potential of chemicals.

3. Background3-1. What is immunotoxicity?
A well-functioning immune system is essential for maintaining the integrity of anorganism. Immune dysregulation can have serious adverse health consequences, ranging
from reduced resistance to infection and neoplasia to allergic and autoimmune
conditions. Many chemicals including environmental contaminants, food additives, andpharmaceutical drugs can affect the immune system, resulting in immune dysregulation.
Currently, the assessment of chemical immunotoxicity relies mainly on animal models
and assays that characterize immunosuppression and sensitization. However, animal
trials have many drawbacks, such as high cost, ethical concerns, and questionable
relevance and inconsistent translatability to risk assessment for humans (Adler et al.,13

2011). In addition, current in vivo models do not always provide a mechanistic
understanding of the data.

3-2. The current status of in vitro immunotoxicity tests
Overcoming some of the drawbacks mentioned above requires the development ofin vitro methods to detect immunotoxicity. A workshop hosted by the European Centre
for the Validation of Alternative Methods in 2003 focused on state-of-the-art in vitro
systems for evaluating immunotoxicity (Galbiati, Mitjans and Corsini, 2010; Gennari etal., 2005; Lankveld et al., 2010), and among the recommendations, a tiered approach
was proposed to assess in vitro immunotoxicity as shown in Fig.1. The proposed tiered
approach would begin with pre-screening for direct immunotoxicity by evaluating
myelotoxicity (Tier 1). Compounds capable of damaging or destroying bone marrow
will most likely have immunotoxic effects. If compounds are not potentially
myelotoxic, they are tested for leukotoxicity (Tier 2). Compounds are then tested for
immunotoxicity using more mechanistically-specific approaches, such as T cell–dependent antibody response (TDAR), lymphocyte proliferation assay, mixed
lymphocyte reaction (MLR), natural killer (NK) cell assay, dendritic cell (DC)maturation assay and human whole-blood cytokine release assay (Tier 3). At present
there is no consensus on which assays to use, or how, and there are no OECD test
guidelines that describe how to detect chemical immunotoxicity in vitro.
Considering the complexity of the immune system and the different underlying
mechanisms of immunotoxicity, it is also clear that one assay alone cannot cover all the14

potential adverse effects of chemicals on the immune system and a larger set of assays,that will cover the spectrum of immunotoxicity, is needed.
Fig. 1. Decision tree approach for in vitro assessment of chemical-induced
immunosuppression

3-3. Predictivity of in vitro immunotoxicity tests
A crucial step in developing an in vitro immunosuppression test involves
determining its predictivity. Determining the predictability of tests requires reference
chemicals that are positive or negative controls based on results obtained by gold
standard analysis or data obtained from the literature. The reference data for thesystemic immunosuppression tests vs target specific immunotoxicity tests should be
different as they should consider the underlying mechanism of action and immune cells
targeted.
In addition, as also applied to non-animal tests for skin sensitization, determining
the predictivity of immunosuppression tests cannot be dependent on a single non-animal
15

alternative assay; rather, it is necessary to develop an IATA approach. Since systemic
immunosuppression likely involves multiple target cells and tissues and/or modes ofaction, it will need to be addressed by multiple adverse outcome pathways (AOPs). As
target-specific effects are more focused, they may be sufficiently explained by a single
AOP using combinations of assays representing different key events (KEs) of the AOP.
Therefore, it may be difficult to reach the predictability goal for a single in vitro
immunosuppression test in validation trials because its predictability will depend on thepercentage of chemicals affecting the specific readout of the test.

3-4. Multi-ImmunoTox Assay (MITA)
Taking into consideration these limitations, our group developed a high-throughput
screening system known as the Multi-ImmunoTox Assay (MITA) to evaluate chemical
immunotoxicity as shown in Fig.2. The MITA utilizes three stable reporter cell lines: 1)
2H4 cells derived from Jurkat cells, expressing stable luciferase green (SLG) regulatedby the interleukin (IL) -2 promoter, stable luciferase orange (SLO) regulated by the
IFN-γ promoter, and stable luciferase red (SLR) regulated by the GAPDH promoter(Saito et al., 2011); 2) THP-G8 cells derived from THP-1 cells, expressing SLO
regulated by the IL-8 promoter and SLR regulated by the GAPDH promoter (Takahashi
et al., 2011); and 3) THP-G1b cells derived from THP-1 cells, expressing SLG
regulated by the IL-1β promoter and SLR regulated by the GAPDH promoter (Kimura
et al., 2014). These four cytokines were selected because IL-2 and IFN-γ are primarily
produced by T cells (adaptive immune cells), whereas IL-8 and IL-1β are primarily
16

produced by monocytes and dendritic cells (innate immune cells). This assay system
can identify the effects of chemicals on IL-2 promotor-driven luciferase activity(IL2LA) and IFN-γ promotor-driven luciferase activity (IFNLA) in 2H4 cells in thepresence of the stimulants phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io)and the effects of chemicals on IL-1 promotor-driven luciferase activity (IL1LA) in
THP-G1b cells and IL-8 promotor-driven luciferase activity (IL8LA) in THP-G8 cells
in the presence of lipopolysaccharide (LPS). We designated the respective componentsof the MITA as the IL-2 Luc assay for detecting the effects of chemicals on IL2LA; theIFN Luc assay for detecting the effects of chemicals on IFNLA; the IL-1 Luc assay for
detecting the effects of chemicals on IL1LA; and the IL-8 Luc assay for detecting theeffects of chemicals on IL8LA.

Fig. 2 The Multi-ImmunoTox Assay (MITA)

17

After establishing the MITA, we first compared the effects of dexamethasone(Dex), cyclosporine (CyA) and tacrolimus (Tac) on the mRNA expression in the three
MITA cell lines with the original cell lines, such as Jurkat cells or THP-1 cells, and withhuman whole-blood cells stimulated with PMA/Io or LPS. The results confirmed that
the MITA correctly reflects changes in mRNA expression in the original cell lines andwhole-blood cells (Kimura et al., 2014).

3-5. The evaluation of immunotoxicity profiles of well-known immunosuppressive
drugs by the MITA
We next evaluated the performance of the MITA by examining
immunosuppressive or immunomodulatory drugs with well-known clinical effects on
the human immune system (Kimura et al., 2014). The results obtained withimmunosuppressive drugs classified by their principal mechanism of action are shownin Table 1. Drug classifications are based on a review by Allison (Allison, 2000).
The MITA demonstrated that Dex significantly suppressed IL-2, IL-1β and IL-8
reporter activity, while CyA and Tac suppressed IL-2 and IFN-γ reporter activity but
had no effect on IL-1β and IL-8 reporter activity. However, the MITA could not detect
the immunosuppressive effects of the alkylating agent cyclophosphamide, of theinhibitors of de novo purine synthesis azathioprine (AZ), mycophenolic acid (MPA) andmizoribine (MZR), or of the inhibitor of pyrimidine and purine synthesis, methotrexate(MT). These data suggest that the MITA correctly evaluates the effects of chemicals on
cytokine expression but cannot detect immunotoxicity associated with the inhibition of18

DNA synthesis and cell division. This drawback has also been reported for other assays,
such as the Human Whole Blood Cytokine Release Assay (HWBCRA; Langezaal et al.,2002) and the Fluorescent Cell Chip (FCC) assay (Wagner et al., 2006). The MITA has
the advantage over other assays in that it can discriminate the effects of chemicals on T
cells from those on macrophages/dendritic cells.

Table 1. The MITA can detect immunomodulatory effects of representative
immunosuppressive drugs

19

S and A indicate drugs that showed statistically significant suppression (S) or augmentation (A) intriplicate experiments for each parameter, while N indicates drugs that did not show significant
effects. 1

3-7. The IL-2 Luc assay plays a principal role in detecting the immunosuppressive
effects of chemicals in the MITA
Table 1 indicated that among the assays included in the MITA, the IL-2 Luc assay
plays a major role in detecting the immunosuppressive effects of chemicals because theIL-2 Luc assay could detect more numbers of immunosuppressive drugs than any othercomponent of the MITA (Kimura et al., 2014; Kimura et al., 2018; Kimura et al., 2020).

3-8. The IL-2 Luc assay cannot detect immunosuppressive effects of drugs which
are dependent on the inhibition of DNA synthesis or anti-proliferative effects
Although the IL-2 Luc assay could detect a significant number of
immunosuppressive drugs such as dexamethasone, calcineurin inhibitors, and several
off-label immunosuppressive drugs, it could not detect immunosuppressive drugs whose
effects are dependent on the inhibition of DNA synthesis or anti-proliferative effects,
such as rapamycin (RPM), mizoribine (MZR), cyclophosphamide (CP), methotrexate(MTX), and mycophonolic acid (MPA) (Kimura et al., 2014; Kimura et al., 2018;
Kimura et al., 2020).

1

Table 1 was adapted from Kimura et al., 201420

3-9. The IL-2 Luc LTT can detect antimitotic effects of chemicals.
Recently, Shao et al. demonstrated that a toxicogenomics approach using Jurkat
cell lines can identify mechanisms underlying the direct immunotoxicity of chemicals,such as endoplasmic reticulum stress, response to oxidative stress, regulation of cell
cycle, and anti-apoptosis (Shao et al., 2014). We therefore speculated whether an assay
could be developed to detect the antimitotc effects of chemicals using 2H4 cell lines.
The 2H4 cell lines simultaneously measure GAPDH promoter-driven luciferase
activity (GAPLA), IL-2LA, and IFNLA (Saito et al., 2011). GAPDH mRNA isubiquitously expressed at moderately abundant levels. It is frequently used as an
endogenous control for quantitative real time polymerase chain reaction because its
expression is constant at different times and after experimental manipulation in someexperimental systems (Edwards and Denhardt, 1985; Mori et al., 2008; Winer et al.,1999). Although there are several reports suggesting that its use as an internal standard
is inappropriate in some cases (Oliveira et al., 1999; Thellin et al., 1999), in general,
there is little within-tissue variation of GAPDH mRNA expression levels (Barber et al.,2005). In addition, in THP-G8 cells that contain the same plasmid as 2H4 (whichcontains the SLR gene driven by the GAPDH promoter), we previously reported asignificant correlation between Inh-GAPLA that is defined as GAPLA of reporter cells
treated with chemicals/GAPLA of untreated cells and the reduction of propidium iodide(PI)-excluding cells, with strengthened correlation with cytotoxicity depending on theculture conditions. In other words, GAPLA shows a good correlation with viable cellnumber and can be used as a marker of cell viability, and in addition, a decrease in
21

GAPLA precedes a decrease in PI-excluding cells (Kimura et al., 2015; Takahashi et al.,2011).
We therefore hypothesized that if 2H4 cells are treated with antimitotic agents for 24 hand then stimulated with PMA/Io, they may reduce GAPLA without suppressing either
IL2LA or IFNLA (Fig. 3). Therefore, we modified the protocol of the IL-2 Luc assay bychanging the incubation period with chemicals from 1 h to 24 h and seeding celldensity. Then, we evaluated the performance of the assay using pharmaceutical drugs
with well-known effects on the immune system. We first examined CyA, AZ, MPA,
MZ, bleomycin, and the control dimethyl sulfoxide (DMSO) by the IL-2 Luc assay withthe modified protocol. As expected, CyA did not affect GAPLA and suppressed theratio of IL2LA/GAPLA, while AZ, MPA, MZ, and bleomycin significantly suppressed
GAPLA and increased the ratio of IL2LA/GAPLA. After we demonstrated that theresults of the luciferase assay corresponded well with the actual change in cell number
and IL-2 production, we constructed an assay to detect the antimitotic effects ofchemicals using 2H4 cells by more precisely defining the protocol and criteria to judge
positive or negative. The IL-2 luciferase assay with this modified protocol is designatedas the IL-2 luciferase leukocyte toxicity test (IL-2 Luc LTT).

22

Fig. 3. The different aspects of immunosuppression detected by the IL-2 Luc assay andthe IL-2 Luc LTT

3-10. The process of validation of the combined assays of the IL-2 Luc assay andthe IL-2 Luc LTT
We have already conducted the validation study of the IL-2 Luc assay (Kimura et al.,2020). In this study, we conducted the validation study for the IL-2 Luc LTT. First, wedemonstrated the within- and between-laboratory reproducibility and then calculated its
performance for detecting anti-mitotic effects and immunosuppressive effects. In
addition, we also evaluated the performance of the combination of the IL-2 Luc assay
and the IL-2 Luc LTT as part of an IATA approach.

23

4. Test method and modification
4-1. IL-2 reporter cell line 2H4
The Jurkat human acute T lymphoblastic leukemia cell line kindly provided byProfessor Kazuo Sugamura, Department of Microbiology, Tohoku University School of
Medicine, was cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO) containing
Antibiotic-Antimycotic (Invitrogen) and 10% HycloneTM fetal calf serum (Thermo
Fisher Scientific, Waltham, MA) (Jurkat growth medium) at 37°C with 5% CO2. The
luciferase reporter assay system was constructed using 3 luciferases that emit green
light (Stable luciferase green; SLG), orange light (Stable luciferase orange; SLO), andred light (Stable luciferase red; SLR) using a single bioluminescent substrate. Namely,
we constructed three luciferase vectors, pSLG-test/Hygr, pSLO-test/Neor, and pSLRtest/Purr, by ligating the BamHI/SacI site of resistant gene vectors containing one ofthree resistant genes, hygromycin (SLG), neomycin (SLO) or puromycin (SLR), SV40
promoter, and HSVtk polyA into luciferase gene vectors, pSLG-test, pSLO-test andpSLR-test (Toyobo, Osaka, Japan), respectively. The activities of the luciferases can bemeasured simultaneously and quantitatively with optical filters. This system can rapidly
and easily monitor the expression of multiple genes (Nakajima et al., 2005; Noguchi etal., 2008).

4-2. Chemical treatment of 2H4 cells and measurement of luciferase activity
The 2H4 cells (1 × 104 cells/50 μL/well) in a 96-well black plate were pretreatedwith different concentrations of individual chemicals for 24 hours. Next, 2H4 cells were24

stimulated with 25 nM of Phorbol 12-myristate 13-acetate (PMA) and 1 mM of
Ionomycin for 6 hours. Two luciferase activities (SLG luciferase activity (SLG-LA) and
SLR luciferase activity (SLR-LA)) were simultaneously determined using a microplatetype luminometer with a multi-color detection system (e.g., Phelios AB-2350 (ATTO),
ARVO (PerkinElmer), Tristar LB941 (Berthold)) and Tripluc Luciferase Assay Reagent(TOYOBO Co., Ltd., Osaka, Japan) according to the manufacturers’ instructions. We
obtained SLG-LA driven by the IL-2 promoter (IL2LA) and SLR-LA driven by GAPDH
promoter (GAPLA) in 2H4 cells. We accounted for the variation in cell number and cell
viability after chemical treatment by normalizing the data for IL2LA (nIL2LA) by
dividing IL2LA with GAPLA in 2H4 cells. In addition, we calculated % suppression and
Inh-GAPLA as follows:% suppression= (1-nIL2LA of 2H4 cells treated with chemicals / nIL2LA of nontreated 2H4 cells) x 100
Inh-GAPLA = GAPLA of 2H4 cells treated with chemicals / GAPLA of untreated cells
Definitions of these terms are provided in Table 2.

25

Table 2. Definition of the parameters in the IL-2 Luc LTT
Abbreviations
GAPLA
IL2LA
IFNLA

Definition
SLR luciferase activity reflecting GAPDH promoter
activity
SLG luciferase activity reflecting IL-2 promoter activity of
2H4 cells
SLO luciferase activity reflecting IFN-g promoter activityof 2H4 cells

nIL2LAIL2LA/GAPLA of 2H4 cells

nIFNLAIFNLA/GAPLA of 2H4 cells% suppression
Inh-GAPLACV05
Min Inh-GAPLA(1-nIL2LA of 2H4 cells treated with chemicals / nIL2LA
of non-treated 2H4 cells) x 100
GAPLA of 2H4 cells treated with chemicals / GAPLA of
untreated cells
The lowest concentration of the chemical at which InhGAPLA becomes < 0.05
The minimum value of Inh-GAPLA of each experiment4-3. Criteria to determine antimitotic effects of chemicals
We set the acceptance criteria and criteria for the Phase I trial of the IL-2 Luc LTT as
follows (IL-2 Luc LTT protocol ver. 1.3):
Acceptance criteria
At the time of each experiment, a control experiment examining nIFNLA of 2H4cells treated with PMA/Io and nIFNLA of non-treated 2H4 cells must be conducted. Then,the fold induction of nIFNLA of 2H4 cells treated with PMA/Ionomycin to nIFNLA of
non-treated 2H4 cells is calculated. If the fold induction is less than 3.0, the results
obtained from these experiments should be rejected.
26

Criteria
The experiments are repeated until 2 consistent antimitotic results, indeterminate
results, or non-antimitotic results are obtained. When 2 consistent results are obtained,
the chemicals are judged as indicated by the obtained consistent results.
In each experiment, if chemicals meet the following criteria described below and give
Min Inh-GAPLA < 0.7, they are judged as antimitotic. Otherwise, they are judged asprovisional non-antimitotic.
The criteria for stimulatory:
1. The mean of % suppression is ≤-35 (stimulatory) with statistical significance. The
statistical significance is judged by its 95% confidence interval.
2. The result shows 2 or more consecutive statistically significant stimulatory data points
or 1 statistically significant stimulatory data point with a trend in which at least 3
consecutive data points decrease in a dose-dependent manner. In the latter case, thetrend can cross 0, if only 1 data point shows the opposite effect without statisticalsignificance.
3. The results are judged using only data obtained at the concentration at which InhGAPLA is ≥0.05.
Of chemicals that are not judged as provisional non-antimitotic, if chemicals do notdemonstrate statistically significant suppressive or stimulatory data points, show Min InhGAPLA > 0.7, and they are insoluble at 10 mg/mL in distilled water, they are judged asindeterminate because they may be not dissolved in the vehicle at the concentration
sufficient to show the effects in the culture medium. Otherwise, they are judged as nonantimitotic.

27

4-4. Bioluminescence system
In a typical dual-reporter assay, firefly luciferase from Photinus pyralis (FLuc) isused as the experimental reporter and Renilla luciferase is used as the internal control
reporter. This internal control reporter connects to a constitutively expressed promoter,such as a virus promoter (e.g., the herpes simplex virus thymidine kinase promoter,simian virus 40 promoter) or a housekeeping gene promoter (e.g., GAPDH, β-actin). This
assay system has been commercialized as the Dual-Luciferase Reporter Assay System by
Promega Corporation. In this system, both luciferase activities are measured sequentiallyfrom single extracts based on their bioluminescent substrate specificity. Firefly luciferase
activity is measured first by adding firefly D-luciferin, then Renilla luciferase activity ismeasured by adding coelenterazine (another name for Renilla luciferin), withconcomitant quenching of firefly luciferase luminescence. Finally, firefly luciferase
activity is normalized by Renilla luciferase activity as the promoter activity (Michelini etal., 2014; Nakajima and Ohmiya, 2010; Roda et al., 2004).
An alternative chemical test using a cell-based assay requires the analysis of a large
number of samples. It is preferable to use an improved assay system whereby gene
expression can be monitored simultaneously. In the MITA, therefore, three kinds of beetle
luciferases that emit either green, orange, or red light with a single bioluminescent
substrate, D-luciferin, are used. Multiple promoter activities are conventionally evaluatedin a one-step reaction by combined use of a commercially available bioluminescentreagent (Tripluc Luciferase Assay Reagent, TOYOBO) and a microplate luminometerequipped with optical filters (Nakajima et al. 2005, 2010).
28

In the IL-2 Luc LTT, the triple-color assay system consists of a green-emitting
luciferase (SLG; λmax = 550 nm) (Ohmiya et al. 2000; Nakajima et al. 2005) for
monitoring IL-2 promoter activity, an orange-emitting luciferase (SLO; λmax = 580 nm)(Viviani et al. 2001; Nakajima et al. 2005) for monitoring IFN-γ promoter activity, and a
red-emitting luciferase (SLR; λmax = 630 nm) (Viviani et al. 1999; Nakajima et al. 2005)
for monitoring internal control promoter (GAPDH) activity. The three luciferases emit
different colors upon reacting with firefly D-luciferin and their luminescence is measured
simultaneously in a one-step reaction by dividing the emission from the assay mixture,measured using optical filters (Nakajima et al. 2005). First, the total relative light units(F0) are measured in the absence of the filters. Then, the F1 and F2 values of light thatpassed through the O56 filter (>560-nm long-pass filter) or the R60 filter (>600-nm longpass filter), respectively, are measured. The three luciferase activities are calculated using
the simultaneous equation shown below by substituting the F0, F1 and F2 values. In thisequation, G, O and R are the activities of the green-, orange- and red-emitting luciferases,
respectively, κGO56, κOO56 and κRO56 are the transmission coefficients for the green-,orange- and red-emitting luciferases of the O56 filter, respectively, and κGR60, κOR60 andκRR60 are the transmission coefficients for the green-, orange- and red-emitting
luciferases of the R60 filter, respectively. The transmission coefficients are simply
estimated using purified recombinant luciferase enzymes (Niwa et al. 2010).

29

1
1  G  F0   1
  
 
 F1  =  κGΟ56 κOΟ56 κRΟ56  O 
 F2   κGR60 κOR60 κRR60  R 
  
 
Luminescence activity is measured using a filtered 96-well microplate luminometer (forexample, Phelios (ATTO, Tokyo, Japan), Tristan 941 (Berthold, Bad Wildbad, Germany),
and the ARVO series (PerkinElmer, Waltham, MA).

30

5. Validation Management Structure
5-1. Validation Management Team (VMT)
Trial Coordinator:

Hajime Kojima (Japanese Center for the Validationof Alternative Methods (JaCVAM), National
Institute of Health Sciences (NIHS), Kawasaki,
Japan), VMT trial coordinator, Chemical supplierand Management of quality control

Lead laboratory:

Setsuya Aiba (Retired, Tohoku University, Miyagi,
Japan), Developer of this assay, Test method,expertise underlying science
Yutaka Kimura (Retired, Tohoku University,
Miyagi, Japan)

International expert members
ECVAM liaison:

Emanuela Corsini (Milan Univ., Italy), Test system
expertise, validation expertise, immunotoxicity
expertise
Erwin L. Roggen (3Rs Management and Consulting
ApS, Denmark), Test system expertise, validation
expertise, immunotoxicity expertise

ICCVAM liaison:

Dori Germolec (Division of Translational
Toxicology (DTT)/NIEHS, USA), Immunotoxicity
expertise

JSIT liaison:

Tomoaki Inoue (Retired, Chugai PharmaceuticalCo., Ltd.), Immunotoxicity expertise

Data management team:Takashi Omori (Department of Clinical Biostatistics
Graduate School of Medicine, Kyoto University
Japan), Data analysis, biostatistics dossier

Chemical Selection Committee

Setsuya Aiba (Retired, Tohoku University)（CSC）

Yutaka Kimura (Retired, Tohoku University)
Hajime Kojima (JaCVAM)
Emanuela Corsini (Milan Univ)
31

Erwin L. Roggen (3Rs Management andConsulting ApS)
Dori Germolec DTT/NIEHS)
Tomoaki Inoue (Retired, Chugai Pharmaceutical Co.,
Ltd.)
Participating Test Facilities

Test Facility 1: Tohoku University, SD: Retired,
Chizu Fujimura
Test Facility 2: AIST, Tsukuba, SD: Rie Yasuno
Test Facility 3: AIST, Takamatsu, SD: Yoshihiro
Nakajima

5-2. Management officeHajime Kojima (JaCVAM)
3-25-26 Tonomachi Kawasaki, Kawasaki, 210-9501
TEL: +81-44-270-6600
h-kojima@nihs.go.jp
5-3. Meetings held
June 12th, 2020 (Web meeting)
The teleconference for the MITA Validation study
Subjects: Proposal of new protocol and future plan for IL-2 Luc assay LTT
VMT members: Corsini, E., Roggen, E., Germolec, D., Inoue, T., Aiba, S., Kimura, Y.,
Omori, T., Nakajima, Y. Yasuno, R., Kojima, H.
July 21st, 2020 (Web meeting)
The teleconference for the MITA Validation study
Subjects: Proposal of new protocol and study plan for IL-2 Luc assay LTT
VMT members: Corsini, E., Roggen, E., Germolec, D., Inoue, T., Aiba, S., Kimura, Y.,
Omori, T., Nakajima, Y., Yasuno, R., Kojima, H.
September 8th, 2020 (Web meeting)
The teleconference for the MITA Validation study
32

Subjects: Results of phase 0 in IL-2 Luc LTT and discussion for protocol.
In house data for IL-2 Luc LTT
Chemical selection for phase I (VMT only)
VMT members: Corsini, E., Roggen, E., Germolec, D., Inoue, T., Aiba, S., Kimura, Y.,
Nakajima, Y., Yasuno, R., Kojima, H.
January 7th, 2021 (Web meeting)
The teleconference for the MITA Validation study
Subjects: Results of phase 1 in IL-2 Luc LTT
VMT members: Corsini, E., Roggen, E., Germolec, D., Inoue, T., Aiba, S., Kimura, Y.,
Omori, T., Nakajima, Y., Yasuno, R., Kojima, H.
July 8th, 2021 (Web meeting)
The teleconference for the MITA Validation study
Subjects: Results of phase 2 in IL-2 Luc LTT
VMT members: Corsini, E., Roggen, E., Germolec, D., Inoue, T., Aiba, S., Kimura, Y.,
Omori, T., Nakajima, Y., Yasuno, R., Kojima, H.

6. Study Design
The validation trial assessed the reliability (reproducibility within and between
laboratories) and relevance (predictive capacity) of the IL-2 Luc LTT with a challenging setof test substances (test items) for which high quality in vitro and in vivo data are available.

The validation study (Phase I and Phase II trials) was conducted by three laboratories,
based on the study design in accordance with study plan (Appendix 1) and using the test
chemicals shown in Table 3. The methods were described above in Section 4 and theprecise protocol is described later in Section 8.

33

7. Test Chemicals
The selection process for the test chemicals for the IL-2 Luc LTT validation study
is described below.
In addition, the chemical categories or physical state and chemical properties (e.g.,solid, liquid) are included in the tables of these test chemicals in order to investigate theapplicability domain.

Table 3.
Trial

Breakdown of the IL-2 Luc LTT validation study
Number of

Number of

test

repetitions

Information obtainedExperiment date

Between-lab

August, 2020

compounds
Phase 0

3 non-coded 1

transferability
Phase I
Phase II

5 coded20 coded3
1

Within & between-lab

September toreproducibility

December, 2020

Between-lab

May to August,reproducibility &2021

predictability

7-1. Basic rules for chemical selection
The selection of test chemicals by the CSC in the VMT was based on publishedpapers on in vivo immunotoxicity tests and validation trials for in vitro alternative
assays on immunotoxicity test methods.

34

7-1-1. Applied selection criteria

information on mode/site of action such as inhibition of cytokine production andantimitotic effectscoverage of a range of relevant chemical classes and product classes

quality and quantity of reference data (in vivo and in vitro)

high-quality data derived from animal and (if available) human trials



information on interspecies variations (for example: variability with regard to
the uptake of chemicals, metabolism, etc.)coverage of a range of toxic effects/potencieschemicals that do not require metabolic activation



appropriate negative and positive controls

physical and chemical properties (feasibility of use in the experimental set-up as
implied by the CAS No.)single chemical entities or formulations of known high purity

availability



cost

In the first phase of the selection procedure, the CSC identified and collected
several existing lists of potential chemical immunotoxicants, such as NTP
IMMUNOTOX, and an EPA candidate list. An extensive literature search was
performed by the CSC to ensure that all the pre-selected chemicals fulfilled theselection criteria described above. In addition, it was decided that at least 20% of the35total chemicals to be tested should provide negative results (i.e., not immunotoxic) toincrease the statistical power of the data analysis.

7-1-2. Chemical acquisition, coding, and distribution
Laboratory transferability, and within- and between-laboratory reproducibility andpredictivity, in all test facilities were assessed using coded chemicals. Coding was
supervised by JaCVAM (Appendixes 2-1 and 2-2), in collaboration with the CSC. The
CSC was responsible for coding and distributing the test chemicals, references, andcontrols for the validation study.

7-1-3. Handling
The chemical master at each test facility received complete information consideredessential regarding the test chemicals (physical state, weight or volume of sample,
specific density for liquid test chemicals, and storage instructions) by JaCVAM.
Moreover, the test facility chemical master stored each chemical under conditions inaccordance with the storage instructions and received sealed safety information such as
the Material Safety Data Sheet (MSDS) describing hazard identification and exposure
control/personal protection for each chemical. The test chemicals were delivered
directly to the study director and the study director was not shown the MSDSs. The
study director was to refer to the MSDSs only in the event of an accident. If the study
director referred to the MSDS, he/she was not to reveal the content of the MSDS to thetest facility technicians.
36

No accidents occurred during the validation study, and all test facilities returned
the MSDSs for the test chemicals to JaCVAM in a sealed envelope upon completion of
the validation study. All test chemicals were disposed of in compliance with the rules
and regulations of the test facilities upon completion of the validation study.

7-2. Pre-validation - Phase 0 trial
Transferability of this assay was checked using three non-coded chemicals:
bleomycin sulfate, dexamethasone and 6-thioguanine (Appendix 3) in three test
facilities, including the lead laboratory. These chemicals were selected by the CSC.

7-3. Validation study - Phase I trial
Within- and between-laboratory reproducibility of this assay was checked using
five coded chemicals in three test facilities, as shown in Table 4 and Appendix 4. Thesechemicals were selected by the CSC based on the in-house dataset of the leadlaboratory. The chemicals were coded by JaCVAM as shown in Table 4 and distributed
to the test facilities. Bleomycin sulfate and cyclosporin were used as positive andnegative controls, respectively.
Table 4. Chemical code list on the phase I trial for IL-2 Luc LTT
No.

Chemical name

CAS No.

Storage

Physicality

Supplier

Lot

1

Mycophenolic acid

24280-93-1

0～10℃

Solid

TCI

CUHFB-QK

2

Indomethacin

53-86-1

Room T.

Solid

SIGMA

059M4169V

3

Cyclosporin A

59865-13-3

2～10℃ (NoFreezing)

Solid

WAKO

15-XJZ-47-1

37

4

5-FU

51-21-8

Room T.

Solid

WAKO

CAH4560

5Mannitol

69-65-8

Room T.

Solid

SIGMA

SLCD7105

7-4. Validation study - Phase II trial
Twenty test chemicals were selected by CSC for between-laboratory
reproducibility as shown in Table 5 and Appendix 5. The chemicals were coded by
JaCVAM as shown in Table 5 and distributed to the test facilities.

Table 5. Chemical code list on the phase II trial for IL-2 Luc LTT
No.
1
3
4

Chemical nameColchicine
Tetrabromobisphenol
A EP
Fludarabine
Nicotinamide (NA)

Supplier
SIGMA

Lot
SLCB8521

Solid

TCI

MHI2H-HT

Room T.
Room T.

Solid
Solid

TCI
SIGMA

QP38F-ER
BCCC51635

556-52-5

2～8℃

LiquidSIGMA

BCCB94526
78
9335-67-1
5392-40-5
147-94-4
33419-42-0
122111-039
154-42-7

Room T.
Room T.
Room T.
Room T.

Solid
Liquid
Solid
Solid

TCI
SIGMA
TCI
TCI

HDBRI-FJSTBJ0146
O6YGN-AFAADNB-PF

Room T.

Solid

TCI

P4UDM-DD

11

Perfluorooctanoic Acid
CitralCytarabine EP
Etoposide
Gemcitabinehydrochloride
6-Thioguanine

Room T.

Solid

TCI

2H2WO-QG

12

Cadmium chloride

10108-64-2

Room T.

Solid

Wako

PEE3332

13
14

Prednisolone
Azathioprine (AZ)

50-24-8
446-86-6

Room T.
-20℃

Solid
Solid

SIGMA
SIGMA

SLCB2470
MKCM1683

15

2-Methyoxyacetic Acid

625-45-6

Room T.

LiquidSIGMA

STBD2084V

16

4-Chloro-ophenylenediamine

95-83-0

Room T.

Solid

SIGMA

14606EDV

17

Dichloroacetic acid79-43-62～8℃

LiquidSIGMA

SHBK0353

Deleterious
Substance

18

nNitrosodimethylamine

62-75-9

Room T.

LiquidTCIVVOVL-BS

Dangerous
Substance

598-55-0

Room T.

Solid

TCI

4P6SO-NB6055-19-2

2～8℃

Solid

SIGMA

MKCL-2547

2

10

19
20

CAS No.
64-86-8

Storage Physicality
Room T.
Solid

79-94-7

Room T.

21679-14-1
98-92-0

Glycidol

Methyl carbamate
Cyclophosphamide
monohydrate

38

RemarkDangerous
Substance

Deleterious
Substance

Dangerous
Substance

39

7-5. Acceptance criteria
The within-laboratory reproducibility for all the test facilities was determined byindependent biostatistical analysis using five coded chemicals, under supervision by the
VMT in the Phase I trial. The proportion of concordance for within-laboratory
reproducibility should be greater than or equal to 80% to be accepted as tentative
acceptance criteria.
Twenty-five coded test items were selected to confirm the between-laboratory
reproducibility in the Phase I and II trials. At the end of the testing, the test facilities
submitted a QC certified copy of the whole study dossier to the trial coordinator (study
plan, raw data, records and data analysis, study report in accordance with the principlesof GLP [OECD, 2018]). The proportion of concordance for between-laboratory
reproducibility should be greater than or equal to 80% to be accepted as acceptancecriteria.

8. Protocols
8-1. Overview of IL-2 Luc LTT
An overview of the IL-2 Luc LTT is shown in Fig. 4. In addition, the final
protocol for the present test (version 001.8) is provided as Appendix 6, 7, and 8 and theprocedures are described in detail below.

40

Fig. 4. Overview of the IL-2 Luc LTT

8-2. Protocol for IL-2 Luc LTT
8-2-1. Reagents and equipment
The following reagents and equipment were used.
For maintenance of the 2H4 cells
・ RPMI-1640 (GIBCO Cat#11875-093, 500 mL)
・ FBS (Biological Industries Cat#04-001-1E Lot: 1524129)
・ 100× concentrated antibiotic and antimycotic (10,000 U/mL of penicillin G, 10,000
mg/mL of streptomycin and 25 mg/mL of amphotericin B in 0.85% saline) (e.g.,
GIBCO Cat#15240-062)
・ Hygromycin B (CAS:31282-04-9, Invitrogen Cat#10687-010)
・ G418 (CAS:108321-42-2, WAKO Cat#074-06801)
・ Puromycin (CAS:58-58-2, InvivoGen Cat#ant-pr-1)

41

For chemical exposure, stimulation, and solvents・ Ionomycin (CAS:56092-82-1, Sigma Cat#I0634)
・ Phorbol 12-myristate 13-acetate (PMA) (CAS:16561-29-8, Sigma Cat#P8139)
・ Ethanol (e.g., Wako Cat#057-00456)
・ Dimethyl sulfoxide (DMSO) (CAS:67-68-5, Sigma Cat#D5879)
・ Distilled water (GIBCO Cat#10977-015)

For measurement of luciferase activity
・

Tripluc® Luciferase Assay Reagent (TOYOBO Cat#MRA-301)

Expendable supplies
・T-75 flask tissue culture treated (e.g., Corning Cat#353136)

・

96 well clear black plate (flat-bottom, for measurement of the luciferase activity, e.g.,
Greiner Bio-one Cat#655090)

・

96 well clear plate (round-bottom, for preparation of chemicals and stimulants)

・96 well assay block, 2 mL (e.g., Costar Cat#3960)

・Seal for 96 well plate (e.g., Perkin Elmer TopSeal-A PLUS Cat#6050185, EXCEL
Scientific SealMate Cat#SM-KIT-SP)

・

Reservoir

・

PipetteEquipment for measurement of luciferase activity
・Measuring device: a microplate-type luminometer with a multi-color detection
system that can accept two optical filterse.g., Phelios AB-2350 (ATTO), ARVO (PerkinElmer), Tristar LB941 (Berthold)・

Optical filter: 560 nm long-pass filter and 600 nm long-pass filter

Others・

Pipetman・

8 channel or 12 channel pipetman (optimized for 10-100 µL)・Plate shaker (for 96-well plate)42

・

CO2 incubator (37°C, 5% CO2)・

Water bath・

Cell counter: hemocytometer, trypan blue

8-2-2. Culture media
Various culture media were used, depending on the purpose of the cell culture.

Table 6. A medium: for maintenance of 2H4 cells (500 mL, stored at 2-8°C)
Final
Reagent

Company

Concentration concentration
in medium

RPMI-1640

GIBCO Cat #11875093

FBS

Requiredamount--

440 mL

-

10 %

50 mL

100×

1×

5 mL

10 mg/mL

0.15 µg/mL

7.5 µL50 mg/mL

300 µg/mL

3 mL

50 mg/mL

200 µg/mL

2 mL

Biological Industries
Cat#04-001-1E
Lot: 1524129

Antibiotic-

e.g., GIBCO Cat

Antimycotic#15240-062

Puromycin

InvivoGen # ant-pr-1

G418

WAKO Cat#07406801

HygromycinB

Invitrogen #10687010

Table 7. B medium: for luciferase assay (30 mL, stored at 2-8°C)
Final
Reagent

Company

Concentration concentration
in medium

RPMI-1640

GIBCO Cat #11875-093

43-

Requiredamount27 mL

FBS

Biological Industries
Cat#04-001-1E

-

10 %

3 mL

Lot: 715004

Table 8. C medium: for thawing 2H4 cells (30 mL, stored at 2-8°C)
Final
Reagent

Company

Concentration concentration
in medium

RPMI-1640

GIBCO Cat #11875-

amount--26.7 mL

-

10 %

3 mL

100×

1×

0.3 mL

093
FBS

Required

Biological Industries
Cat#04-001-1E
Lot: 1524129

Antibiotic-

e.g., GIBCO Cat

Antimycotic#15240-062

8-2-3. Preparation of stimulant for 2H4 cells
Table 9-1. Phorbol 12-myristate 13-acetate (PMA)
Reagent

Company

Phorbol 12-myristate 13-acetate

Sigma #P8139

Concentration of

Finalstock solution

concentration

2 mM

25 nM

(PMA)
DMSO

Sigma #D5789

Dissolve 1 mg PMA using DMSO 811 µL, dispend at 5 µL/tube and store at freezer at 30°C. Use these stocks within 6 months after dissolution.

44

Table 9-2. Ionomycin
Reagent

Company

Ionomycin

Sigma # I0634

Ethanol

Wako #057-00456

Concentration of the

Finalstock solution

concentration

2 mM

1 µM

Dissolve 1mg Ionomycin using ethanol 669.3 µL, dispend at 15 µL/tube and store at
freezer at -30°C. Use these stocks within 6 months after dissolution.
8-2-4. Thawing of 2H4 cells
Pre-warm 9 mL of C medium in a 15 mL polypropylene conical tube in a 37°C
water bath (for centrifugation) and 15 mL of A medium in a T-75 flask at 37°C in a 5%
CO2 incubator (for culture).
Thaw frozen cells (5 × 106 cells/0.5 mL of freezing medium) in a 37°C waterbath, then add to a 15 mL polypropylene conical tube containing 9 mL of pre-warmed C
medium. Centrifuge the tube at 120-350 × g at room temperature for 5 min, discard thesupernatant, and resuspend the cells in 15 mL of pre-warmed C medium in a T-75 flask.
Cells are incubated at 37°C, 5% CO2.

8-2-5. Maintenance of 2H4 cells
Pre-warm the A medium in a T-75 Flask at 37°C in a 5% CO2 incubator. The culture
medium should be changed to the A medium 3 or 4 days after thawing. At that time, countthe number of cells, centrifuge the tube at 120-350 x g at room temperature for 5 min,discard the supernatant, and resuspend in pre-warmed the A medium in a T-75 Flask.
Cells are passaged at 1~3x105/mL and incubated at 37°C, 5% CO2.
45

The interval between subcultures should be 3~4 days. Cells can be used between one
and six weeks after thawing.
The lead laboratory has examined how long 2H4 cells could be cultured without
losing their reactivity to PMA/Io. 2H4 cells maintained their response to PMA/Io up to
16 weeks or 35 passages.

8-2-6. Preparation of cells for assay
Cells should be passaged 3-4 days before the assay.
Use cells between 1 and 6 weeks after thawing.
Pre-warm the B medium in a 37°C water bath. Count the number of cells and collectthe number of cells needed (1.0 × 106 cells for two chemicals are required, but to havesome leeway, 1.5 × 106 cells for two chemicals should be prepared), centrifuge the tube
at 120-350 x g, 5 min. Resuspend in the pre-warmed B medium at a cell density of 2 ×
105/mL. Transfer the cell suspension to a reservoir (Thermo Scientific) and add 50 μL ofcell suspension to each well of a 96-well clear black plate (flat bottom) using an 8 channel
or 12 channel PipetmanTM (Gison, Inc., Middleton, WI, USA). Each well of the 96-wellplate should contain 1 × 104 2H4 cells + 50 μL of B medium.

8-2-7. Preparation of chemicals and cell treatment with chemicals
Dissolve the chemical first in distilled water. Weigh 10 mg of the test chemical in a
volumetric flask and add distilled water up to 1 mL. If the chemical is soluble at
10 mg/mL, use 10 mg/mL solution for the stock solution.
46

If the chemical is not soluble at 10 mg/mL in water, the chemical should be dissolvedin DMSO at 200 mg/mL. For example, weigh 200 mg of the test chemical in volumetricflask and add DMSO up to 1 mL. If the chemical does not dissolve in DMSO at
200 mg/mL, use the highest concentration possible after diluting with DMSO at a dilution
factor of 2.
For chemicals that are difficult to obtain in sufficient quantities, prepare the highest
concentration possible instead of 10 mg/mL distilled water. If the chemical is not solubleat 10 mg/mL, prepare the highest concentration possible in DMSO.
Sonication and vortexing may be used if needed, with an attempt to dissolve thechemical for at least 5 minutes. Solubility should be confirmed by the absence of
precipitation following centrifugation at 15,000 rpm (≈20,000 x g) for 5 min. The
chemical should be used within 4 hours after being dissolved in distilled water or DMSO.
(Fig. 5).

Fig. 5. Dissolution by solvent47

8-2-8. Dilution of chemicals
For water soluble chemicals, 10 serial dilutions were conducted using distilled water,
diluting by a factor of 2, in the 1st, 2nd, 3rd, and 4th experiment. For water insoluble
chemicals, 10 serial dilutions were conducted using DMSO as the solvent, diluting by afactor of 2 in the 1st, 2nd, 3rd, and 4th experiments. The solutions are further diluted andadded to 2H4 cells in a 96 well plate. After 24-hour incubation at 37°C in a 5% CO2
incubator, 2H4 cells were added to 10 µL of PMA/ionomycin solution and incubatedagain at 37°C in a 5% CO2 incubator for 6 hours.

8-2-9. Measurements
After incubation with the chemical and PMA/ionomycin for 6 hours at 37°C in
a 5% CO2 incubator, 100 µL of pre-warmed Tripluc is added to each well in the platecontaining reference samples using a pipette and the plate is shaken for 10 minutes atroom temperature using a plate shaker. Surface bubbles are removed if present andbioluminescence in each well is measured using a microplate-type luminometer with amulti-color detection system (Phelios; Atto Co., Tokyo, Japan) for 3 seconds each in theabsence (F0) and presence (F1, F2) of the optical filter. The F0, F1 and F2 data (values
are expressed as counts) are processed using an Excel-based data sheet (Appendix 9).
IL2LA, IFNLA and GAPLA are calculated for each well based on the algorithm tocalculate IL2LA, IFNLA and GAPLA from the raw luminescence data reported
previously (Nakajima et al., 2005; Noguchi et al., 2008). In addition to being used to
calculate IL2LA, IFNLA and GAPLA, this data sheet can automatically generate final48

graphs showing the correlation between % suppression and the concentration ofchemicals, and between Inh-GAPLA and the concentration of the chemical.

8-2-10. Luminometer apparatus
Multi-color detection systems such as microplate luminometers are available(e.g., Phelios (ATTO, Tokyo, Japan), Tristan 941 (Berthold, Bad Wildbad, Germany), and
the ARVO series (PerkinElmer, Waltham, MA)). The luminometer detectors must havehigh sensitivity (especially for the red region) and low background noise, and are usuallyequipped with optical filters such as sharp-cut (long-pass) filters or band-pass filters. The
transmission coefficients for these filters against each luciferase must be estimated prior
to initiating the experiments because the coefficients are dependent on the luminometer
due to lot-to-lot variations in detectors.

8-2-11. Positive and negative controls
In each experimental set, bleomycin sulfate and dexamethasone are used as positive
and negative controls, respectively.

8-2-12. Calculation and definition of parameters for IL-2 Luc LTT
In the IL-2 Luc LTT, nIL2LA was defined to represent IL-2 promoter-driven SLG
luciferase activity (IL2LA) normalized by the SLR luciferase activity (GAPLA). The
inhibition of GAPLA (Inh-GAPLA) was determined by dividing the GAPLA for 2H4

49

treated with chemicals with the GAPLA for non-treated 2H4. Percent suppression
reflects the effect of chemicals on the IL-2 promoter (Table 10).
Table 10. Abbreviations used in the IL-2 Luc LTT protocol
Abbreviations
GAPLA
IL2LA
IFNLA

Definition
SLR luciferase activity reflecting GAPDH promoter activity
SLG luciferase activity reflecting IL-2 promoter activity of
2H4 cells
SLO luciferase activity reflecting IFN-γ promoter activity of
2H4 cells

nIL2LAIL2LA/GAPLA of 2H4 cells

nIFNLAIFNLA/GAPLA of 2H4 cells% suppression
Inh-GAPLA
CV05(1-nIL2LA of 2H4 cells treated with chemicals / nIL2LA of nontreated 2H4 cells) x 100
GAPLA of 2H4 cells treated with chemicals /GAPLA of
untreated cells
The lowest concentration of the chemical at which Inh-GAPLA
becomes < 0.05

Min Inh-GAPLA The minimum value of Inh-GAPLA of each experiment8-2-13. Acceptance criteria
The following acceptance criteria should be satisfied when using the IL-2 Luc LTT
method.
•For each set of experiments, a control experiment examining nIFNLA of 2H4 cells
treated with PMA/Io and nIFNLA of non-treated 2H4 cells must be conducted. Then,the fold induction of nIFNLA of 2H4 cells treated with PMA/Ionomycin to nIFNLA

50

of non-treated 2H4 cells is calculated. If the fold induction is less than 3.0, the results
obtained from these experiments should be rejected.
8-2-14. Criteria
The experiments are repeated until 2 consistent antimitotic results, indeterminate
results, or non-antimitotic results are obtained. When 2 consistent results are obtained,
the chemicals are judged as indicated by the obtained consistent results.
In each experiment, if chemicals meet the following criteria described below
and give Min Inh-GAPLA < 0.7, they are judged as antimitotic. Otherwise, they are
judged as provisional non-antimitotic (Fig. 6).
The criteria for antimitotic:
1. The mean of % suppression is ≤-35 (stimulatory) with statistical significance. The
statistical significance is judged by its 95 % confidence interval.
2. The result shows 2 or more consecutive statistically significant stimulatory data points
or 1 statistically significant stimulatory data point with a trend in which at least 3
consecutive data points decrease in a concentration-dependent manner. In the latter
case, the trend can cross 0, if only 1 data point shows the opposite effect without
statistical significance.
3. The results are judged using only data obtained at the concentration at which InhGAPLA is ≥0.05.

Of chemicals that are judged as provisional non-antimitotic, if chemicals do not give
statistically significant suppressive or stimulatory data points, give Min Inh-GAPLA >0.7, and they are insoluble at 10 mg/mL in distilled water, they are judged asindeterminate because they may be not dissolved in the vehicle at the concentration51

sufficient to show the effects in the culture medium. Otherwise, they are judged as nonantimitotic.

Fig. 6 Criteria of IL-2 Luc LTT

8-3. Data collection
8-3-1. Operating procedure
Details of the operating procedure for this assay are described in protocol version
001.4 (Appendix 6). Versions of the protocols were updated during the validation trials,
but the descriptions of the operating procedures described in these protocols were thesame for the two validation trials.

52

8-3-2. Chemicals
For the Phase I trial, in which the main aim was to evaluate intra- and inter-laboratory
reliability, a total of 15 coded chemicals were tested in three rounds of five chemicals
each distributed to three laboratories. Different codes were used in the rounds and thus
the technician in each laboratory could not identify the chemicals. For the Phase II trial,in which the main aim was to evaluate inter-laboratory reliability, 20 coded chemicals
were distributed.
In this document, the chemicals were re-coded. The round is indicated by a suffix suchas P101_R1 for the first chemical of the first round of the Phase I trial: P1 means Phase
I; 01 means the first chemical; _R1 means first round.

8-3-3. Data handling
The Excel data sheet developed for this study was distributed to the laboratories. Data
files were received from each of the three laboratories.
JaCVAM, provided the files listing the chemical codes for the five distributed
chemicals in the Phase I trial, and 20 chemicals in the Phase II trial.
For data analysis, these files were combined and datasets were constructed. SAS ver.
9.4 and Microsoft Excel were used for the data analyses described in this report.
Since Excel data sheets can display a concentration-response plot for % suppression
with its 95% confidence interval, we could judge “Stimulatory” or “Negative” for eachexperiment from the plot (Appendix 9).

53

8-3-4. Index from each experiment and decision criteria for judgment
The j-th repetition (j = 1 to 4) of the i-th concentration (i = 0 to 11) was measured for
IL2LA and GAPLA. The normalized IL2LA is referred as nIL2LA and is defined as
nIL2LAij = IL2LAij/GAPLAij.
This is the basic unit of measurement in this assay.

8-3-4-1. % suppression% suppression is an index for the averaged nIL2LA for the repetition using the i-th
concentration compared with at 0 concentration and is the primary measure in this assay.
Suppression (%) is described by the following formula:1� � ∑i nIL2LAij% Suppressioni = �1 − 4 1� �𝑛𝑛𝑛𝑛𝑛𝑛2LA0j
4

� × 100(1)

The lead laboratory has proposed that -35 of the value suggests stimulatory for a tested
chemical. This value is based on the investigation of the historical data of the leadlaboratory. Data management team used this value through all the phases of the present
validation study.
The primary outcome measure, % suppression, is basically the ratio of twoarithmetic means of nIL2LA, as shown in the equation below. The 95% confidence
interval (95% CI) for % suppression for the i-th concentration can be estimated.
The lower limit of the 95% CI above 0 is interpreted as the nIL2LA of the i-th
concentration being statistically significantly greater than at 0 concentration.

54

There are several ways to construct the 95% CI. We used the method known as the
Delta method. This 95% confidence interval is obtained from the following formula:sd2i% suppression ±100 × �𝑧𝑧0.975 × �mean
2 +0

mean2i ×sd20
mean40�,

where meani is the mean of nIL2LA at the i-th concentration, mean0 is the mean ofnIL2LA at 0 concentration, sdi is the standard deviation of nIL2LA at the i-th
concentration, and sd0 is the standard deviation of nIL2LA at 0 concentration. z0.975 is

the 97.5 percentile of the standard normal distribution.

8-3-4-2. Inh-GAPLA
Inh-GAPLA is a ratio of the averaged GAPLA for the repetition of the i-th
concentration compared with 0 concentration, and is written as11

Inh − GAPLAi = �4� ∑i GAPLAij / �4� ∑i GAPLA0j .

Since GAPLA is the denominator of the nIL2LA, an extremely small GAPLA valuecauses a large variation in nIL2LA. Therefore, the i-th % suppression value with anextremely small value of Inh-GAPLA might result in poor precision.

8-3-4-3. Judgment for “antimitotic” or “non-antimitotic” in each experiment
In each experiment, if chemicals meet the criteria described below and give Min InhGAPLA < 0.7, they are judged as antimitotic. Otherwise, they are judged as provisional
non-antimitotic (Fig. 6).
The criteria for stimulatory:55

1. The mean of % suppression is ≤-35 (stimulatory) with statistical significance. The
statistical significance is judged by its 95% confidence interval.
2. The result shows 2 or more consecutive statistically significant stimulatory data points
or 1 statistically significant stimulatory data point with a trend in which at least 3
consecutive data points decrease in a concentration-dependent manner. In the latter
case, the trend can cross 0, as long as only 1 data point shows the opposite effectwithout statistical significance.
3. The results are judged using only data obtained at the concentration at which InhGAPLA is ≥0.05.
Of chemicals that are judged as provisional non-antimitotic, if chemicals do notdemonstrate statistically significant suppressive or stimulatory data points, show Min
Inh-GAPLA > 0.7, and they are insoluble at 10 mg/mL in distilled water, they are
judged as indeterminate because they may be not dissolved in the vehicle at theconcentration sufficient to show the effects in the culture medium. Otherwise, they
are judged as non-antimitotic.

8-3-4-4. Final judgment for “antimitotic” or “antimitotic” using this assay
In this assay, “antimitotic” or “non-antimitotic” is defined as the case in which there
are two identical judgments in a set of experiments.

8-3-5. Reliability
8-3-5-1. Within-laboratory reproducibility for five common chemicals
56

Within-laboratory reproducibility was determined by whether or not threeexperimental sets for each chemical by each laboratory were concordant. The
concordance rate was then calculated as the proportion of concordance for each laboratory.
The concordance rate for within-laboratory reproducibility was based on the results ofthree sets.
To summarize, the concordance rate for within-laboratory reproducibility from the threelaboratories was used to calculate the averaged concordance rate.

8-3-5-2 Between-laboratory reproducibility
Between-laboratory reproducibility was determined using the results from the final
judgment from the three laboratories for 25 chemicals, this is, five chemicals in the Phase
I trial and 20 chemicals in the Phase II trial. These judgements were tabulated, and thenthe concordance rate was calculated as a proportion of the concordance in each laboratory.
To summarize, the concordance rate for between-laboratory reproducibility from thethree laboratories was used to calculate the averaged concordance rate.

8-3-6. Predictivity
8-3-6-1. Definition of concordance, sensitivity, and specificity
The concordance, sensitivity, and specificity were estimated as the indices of predictivity.
These indices were estimated using the frequency results obtained from the 2 × 2
contingency table for leukocyte toxicity. The definitions of these indices are summarized57

in Table 11 below. This calculation was based on the results decided by the majority forthe between-laboratory results for each chemical.
Table 11. Definition of concordance, sensitivity, and specificity
Judgment from IL-2 Luc LTT

Chemical category

Total

Positive

Negative

Positive

a

b

a+b

Negative

c

d

c+d

Total

a+c

b+d

N

Sensitivity = 100 x a/(a+c) or the ability to predict a positive result
Specificity = 100 x d/(b+d) or the ability to predict a negative result
Accuracy = 100 x (a+d)/N

8-4. Quality assurance
Assays and quality assurance were carried out in the spirit of GLP, as not all theparticipating laboratories routinely worked under GLP certification. The participating
laboratories conducted the experiments in accordance with the protocol provided by the
VMT. All raw data and data analysis sheets were pre-checked for quality by eachlaboratory and were then reviewed by the VMT quality assurance team. The results
accurately reflected the raw data.

9. Results
We conducted Phase I and II trials in this validation study. The assay procedure andcriteria used to judge antimitotic in the validation trials are summarized in Fig. 7.
58

Fig. 7. The progress of the validation study for the IL-2 Luc LTT.

9-1. Final criteria for Phase I trial
9-1-1. Acceptance criteria
The following acceptance criteria should be satisfied when using the IL-2 Luc
LTT.
•

For each set of experiments, a control experiment examining nIFNLA of 2H4 cells
treated with PMA/Io and nIFNLA of non-treated 2H4 cells must be conducted. Then,the fold induction of nIFNLA of 2H4 cells treated with PMA/Ionomycin to nIFNLA
of non-treated 2H4 cells is calculated. If the fold induction is less than 3.0, the results
obtained from these experiments should be rejected.

59

9-1-2. Criteria
The experiments are repeated until 2 consistent antimitotic results, indeterminate
results, or non-antimitotic results are obtained. When 2 consistent results are obtained,
the chemicals are judged as indicated by the obtained consistent results.
In each experiment, if chemicals meet the following criteria described below andhave Min Inh-GAPLA < 0.7, they are judged as antimitotic. Otherwise, they are judged
as provisional non-antimitotic.
The criteria for stimulatory:
1. The mean of % suppression is ≤-35 (stimulatory) with statistical significance. The
statistical significance is judged by its 95% confidence interval.
2. The result shows 2 or more consecutive statistically significant stimulatory data points
or 1 statistically significant stimulatory data point with a trend in which at least 3
consecutive data points decrease in a concentration-dependent manner. In the latter
case, the trend can cross 0, if only 1 data point shows the opposite effect without
statistical significance.
3. The results are judged using only data obtained at the concentration at which InhGAPLA is ≥0.05.
Of chemicals that are judged as provisional non-antimitotic, if chemicals do notdemonstrate statistically significant suppressive or stimulatory data points, show Min InhGAPLA > 0.7, and they are insoluble at 10 mg/mL in distilled water, they are judged asindeterminate because they may be not dissolved in the vehicle at the concentration60

sufficient to show the effects in the culture medium. Otherwise, they are judged as nonantimitotic.

9-1-3. Predictivity
The IL-2 Luc LTT is aimed to detect antimitotic effects of chemicals. We firstexamined the performance of the IL-2 Luc LTT for detecting antimitotic effects ofchemicals. Then, we examined its performance for immunosuppressive effects because
it has a potential to detect immunosuppression caused by antimitotic effects. To
determine the predictivity of the immunotoxicity test, reference data that aid positive or
negative judgments are essential. Since no such reference data are currently available todetermine the predictability of the IL-2 Luc LTT, we prepared reference data. To makethe reference data for immunosuppression, we referred to the rationale for
immunotoxicity classification proposed by Luster et al (Luster, 1998) in which they
conducted statistical evaluation of data from a screening battery using a tier approach todetect potential immunotoxic compounds in mice (Luster et al., 1992). According totheir rationale, a positive reference chemical would either produce a significant
concentration-dependent effect in the immune test or significantly alter two or moreimmune test results at the highest concentration of the chemical tested. Chemicals wereclassified based on the results obtained in 12 immune tests according to this rationale
and found a significant correlation between the judgment of immunotoxic chemicals
and host resistance (Luster et al., 1993). Therefore, using this rationale, we classified
chemicals as described in our previous publication (Kimura et al., 2020).
61

Briefly, we first surveyed the literature, collected the following eight endpoints
regarding each chemical used in the study (Table 12), and generated reference data for
their immunosuppressive profiles used in the validation study (Appendix 12). The
references of the literature that Appendix 12 referred to were shown in Appendix 13.
Then, we classified a chemical as an immunosuppressive chemical if it meets Criterion
1 or classified a chemical as anti-mitotic if it meets Criterion 2 (Table 13). The
summarized immunotoxicity information and the classifications of the chemicals arealso shown in Appendix 12.
Then, by comparing the results of the IL-2 Luc LTT (positive or no effect) with theclassification of the chemicals (immunosuppressive or non-immunosuppressive), we
calculated the accuracy, sensitivity, and specificity of the IL-2 Luc LTT in thevalidation study.
Table 12. Immunotoxicological data obtained from literature.
Endpoint

Information

Endpoint 1

Decreased antibody response

Endpoint 2

Myelosuppressive or antimitotic

Endpoint 3

Decreased LPS response in vivo, ex vivo, or in vitro

Endpoint 4

Suppressed DHR

Endpoint 5

Suppressed host resistanceEndpoint 6

The NTP data or Tox 21 data indicate immunotoxicity of chemicals

Endpoint 7

Decreased thymus weight

Endpoint 8

Increased or decreased IL-2 mRNA expression or protein production by T
cells in ex vivo.

Endpoint 9

Increased or decreased IL-2 mRNA expression or protein production by T
cells in vitro.

62

Table 13. Criteria to classify immunotoxic chemicals
Criterion

Definition

Criterion 1 (systemic immunotoxicity)

Satisfy one of Endpoints 1 to 9

Criterion 2 (antimitotic effect)

Satisfy Endpoint 2

9-2. Phase 0 trial (for technical transfer)
The preliminary test trial (Phase 0) was performed by the participating
laboratories following explicit explanations of “IL-2 Luc leukocyte toxicity test (IL-2 Luc
LTT) protocol ver.001.2” by the lead laboratory, Tohoku University. Three laboratories
participated in the Phase 0 trial of the IL-2 Luc LTT using the three open-labeledchemicals, bleomycin sulfate, dexamethasone, and 6-thioguanine, and conducted one set(three experiments) for each chemical. The response patterns for the three chemicals weresimilar among the three laboratories. Based on these results, the VMT judged thattechnical and protocol transfer of the IL-2 Luc LTT was acceptable.

9-3. Phase I trial (for within- and between-laboratory reproducibility)
9-3-1. Test conditions
A total of five coded chemicals were evaluated by three experimental sets in the
Phase I trial based on “IL-2 Luc leukocyte toxicity test (IL-2 Luc LTT) protocol
ver.001.3”.
In each experimental set, initially two experiments are conducted. If the results areconcordant chemical will be classified accordingly, if discordant an additional
experiment is conducted. In case of null experiments, they must be repeated.
63

Chemicals that satisfied the criteria were judged as positive. Chemicals thatprovided two positive results were judged as antimitotic.

9-3-2. Within-laboratory variation assessments in Phase I trial
The complete results of the Phase I trial are shown in Table 14 and Appendix 10.
The within-laboratory reproducibility was 100.0% (15/15), 100% (15/15), and 100.0%(15/15) in Labs A, B, and C, respectively. The average reproducibility was thus 100.0%(15/15).
9-3-3. Between-laboratory variation assessments in Phase I trial
The between-laboratory reproducibility was also 100.0% (15/15).

Table 14. Results of Phase I trial
Chemical

CAS

Set

Lab A

Lab B

Lab C

Concordance

Mycophenolic

24280-

acid

93-1

1st

Antimit Antimit Antimit
otic

2nd

53-86-1

1st

Cyclosporin A

1st

otic

otic

otic

1

otic

Antimit Antimit Antimit
otic

3rd

otic

Antimit Antimit Antimit
otic

2nd

otic

Antimit Antimit Antimit
otic

Indomethacin

oticAntimit Antimit Antimit
otic

3rd

otic

1

otic

otic

Antimit Antimit Antimit
otic

otic

otic

Non

Non

Non

64

1

5-Fluorouracil

59865-

2nd

Non

Non

Non

13-3

3rd

Non

Non

Non

51-21-8

1st

Antimit Antimit Antimit
otic

2nd

otic

Antimit Antimit Antimit
otic

3rd

otic
otic

otic

Antimit Antimit Antimit
otic

otic

otic

1st

Non

Non

Non

2nd

Non

Non

Non

3rd

Non

Non

Non

Within-

100

100

100

laboratory(15/15)(15/15)(15/15)Mannitol

69-65-8

1

reproducibility

Average(%)

100 (15/15)

1100 (5/5)

Betweenlaboratory
reproducibility(%) (based on
majority)

9-4. Phase II trial (for between-laboratory reproducibility and predictivity)
9-4-1. Test conditions
The Phase II trial to examine between-laboratory reproducibility and predictivity
was conducted using a total of 20 coded chemicals and evaluated using one experiment
set based on “IL-2 Luc leukocyte toxicity test (IL-2 Luc LTT) protocol ver.001.4”.

65

9-4-2. Between-laboratory variation assessments in Phase II trial
The complete results of the Phase II trial are shown in Table 15 and Appendix 11.
The between-laboratory reproducibility was 90% (18/20).
Table 15. Results of Phase II trial
ChemicalColchicine

CAS64-86-8

Lab A

79-94-7

AEP
Fludarabinetic

Based onConcor-majority

dance

otic

tic

otic

1

otic

tic

otic

tic

Nicotinamide

98-92-0

Non

Non

Non

Non

Glycidol

556-52-5

Antimit Antimito Antimit Antimito

Perfluorooctanoic Acid

335-67-1

tic

5392-40-5

tic

Antimit Antimito
otic

Cytarabine EP

147-94-4
33419-420

Gemcitabine

122111-hydrochloride

03-9

6-Thioguanine

154-42-7

tic

10108-642

Non

Antimito

otic

tic

otic

tic

otic
otic

tic

66

0

otic
otic

1
1
1

tic
1

tic

Antimit Antimito Antimit Antimito
otic

1

tic

Antimit Antimito Antimit Antimito
tic

1

tic

Antimit Antimito Antimit Antimito
otic

1

tic

Antimit Antimito Antimit Antimito

otic
Cadmium chloridetic

Antimit Antimito Antimit Antimito
otic

Etoposide

otic

tic

1

tic

Antimit Antimito Antimit Antimito
otic

Citral

otic

1

tic

Antimit Antimito Antimit Antimito

otic

1

tic

Antimit Antimito Antimit Antimito
otic

21679-14-

Lab C

Antimit Antimito Antimit Antimito
otic

Tetrabromobisphenol

Lab B

tic

1

Prednisolone

50-24-8

Antimit Antimito Antimit Antimito
otic

tic

otic

tic

1

Azathioprine

446-86-6

Non

Non

Non

Non

1

2-Methyoxyacetic Acid

625-45-6

Non

Non

Non

Non

1

4-Chloro-o-

95-83-0Non

Antimito

Non

Non

0

phenylenediamine

tic

Dichloroacetic acid79-43-6

Non

Non

Non

Non

1

n-Nitrosodimethylamine

62-75-9

Non

Non

Non

Non

1

Methyl carbamate

598-55-0

Non

Non

Non

Non

1

Cyclophosphamide

6055-19-2

Non

Non

Non

Non

1

monohydrate
Between-laboratory

90 (18/20)

reproducibility (%)

9-5. Quality assurance (Appendix 16)
No accidents occurred during the validation study, and all test facilities returned
the MSDSs for the test chemicals to JaCVAM in sealed envelopes upon completion of
the validation study. All test chemicals were disposed of in compliance with the rules
and regulations of the test facilities upon completion of the validation study.
All the records (data sheets and record sheets) from the participating laboratories
were checked by Dr. Takashi Omori, Kobe Univ. and JaCVAM. The check lists areavailable at Appendix 17-1 and 17-2. The record sheets include “Chemical records,
Solubility test records, Cell culture records and Testing records”. The records are total
more than 300 pages and available at JaCVAM website (http://
http://www.jacvam.jp/validation08-login.html). Testing performed as part of thevalidation study was carried out in non-GLP laboratories in accordance with the67principles of GLP (OECD, 1998) and necessarily include, without being limited to, theuse of protocol and adequate recording of data as well as suitable reporting of results
and archival record keeping.
We found several insufficient descriptions in the culture of the cells, the preparation andapplication of test chemicals and data sheets, because a few record sheets wereincomplete. Corrected record sheets should be provided before the completion of thevalidation study and test developers and an independent organization should review
them in advance. The results accurately reflect the raw data and the incomplete
descriptions did not influence the results.
The following concerns were noted in the validation trials. The QC team requested torevise the protocol after the validation study.
1)

The measurements of luciferase activity were done up to 33oC in all thelaboratories. Although the record sheet required a record of actual temperature,
there is no description of experimental temperature in protocol. The QC teamrequested to describe/recommends describing the measured temperature in the
protocol if it affects the test results.

2)

The culture period after addition of PMA/ionomycin is 6 hr in the protocol.
However, the culture period in a laboratory was done up to 7 hr. The QC teamrequested to revise/recommends revising the protocol based on scientific data.

3)

The experiment’s number of positive and negative controls are not fixed in the
protocol. The QC team requested to describe/recommends describing that the68control tests should be done at the same time as each experiment in the protocol todemonstrate that each experiment was done adequately.

The QC team carefully checked the other results and judged all data to be within
acceptable ranges. The QC team recommended to JaCVAM that the validation study
performed with GLP laboratories should be move forward.

9-6. Combined results of Phase I and II trials (for between- and within- laboratory
reproducibility)
9-6-1. Test conditions
The within- and between-laboratory reproducibility, and the predictivity of the IL-2 Luc
LTT, were evaluated using all the results from the phases I and II trials.

9-6-2. Between-laboratory variation assessments from Phase I and II trials(Table 16)
To further evaluate the between-laboratory reproducibility, the results from thePhase I and II trials were combined. The reproducibility for the combined results was
92.0% (23/25), similar to that of the Phase II trial alone.

69

Table 16. Combined results of Phase I and II trials
Chemical

CAS

Lab A

Lab B

Lab C

24280

Antimito

Antimitot Antimitot

-93-1

tic/

Concord-ance

Phase I
Mycophenolic acid

Antimitotic/
Antimitotic
Indomethacin

53-

Antimito

86-1

tic/
Antimitotic/
Antimito

Cyclosporin A
5-Fluorouracil

ic/

ic/

Antimitot Antimitot
ic/

ic/

Antimitot Antimitot
ic

ic

Antimitot Antimitot
ic/

ic/

Antimitot Antimitot
ic/

ic/

ic

ic

59865

Non/NonNon/NonNon/Non

-13-3

/Non

/Non

/Non

51-

Antimito

21-8

tic/
tic/
Antimito1

Antimitot Antimitot
ic/

ic/

Antimitot Antimitot
ic/

ic/

1

Antimitot Antimitot

tic

ic

ic

69-

Non/NonNon/NonNon/Non

65-8

/Non

/Non

/Non

64-

Antimito

86-8

tic

Tetrabromobisphenol A

79-

Antimito

EP

94-7

tic

Mannitol1

Antimitot Antimitot

tic

Antimito1

1

Phase IIColchicine

70

Antimitot Antimitot
ic

ic

Antimitot Antimitot
ic

ic

1
1

Fludarabine
Nicotinamide
Glycidol
Perfluorooctanoic Acid
CitralCytarabine EP
Etoposide
Gemcitabinehydrochloride
6-Thioguanine
Cadmium chloride
Prednisolone
Azathioprine
2-Methyoxyacetic Acid

21679

Antimito

-14-1

tic

ic

ic

Non

Non

Non

9892-0556-

Antimito

52-5

tic

335-

Antimito

67-1

tic

ic

5392-

Antimito

Antimitot

40-5

tic

ic

147-

Antimito

94-4

tic

33419

Antimito

-42-0

tic

12211
1-039

Antimitotic

Antimitot Antimitot
ic

ic

Antimitot Antimitot
ic
Non

Antimitot Antimitot
ic

ic

Antimitot Antimitot
ic

ic

Antimitot Antimitot
ic

ic

1
1
1
0
1
1

1

Antimito

42-7

tic

10108

Antimito

-64-2

tic

50-

Antimito

24-8

tic

ic

ic

Non

Non

Non

1

Non

Non

Non

1

Non

0

Non

1

44686-662545-695-

phenylenediamine

83-0
7943-6

Non
Non

71

Antimitot Antimitot

1

154-

4-Chloro-o-

Dichloroacetic acid

Antimitot Antimitot

ic

ic

Antimitot Antimitot
ic

ic

Antimitot Antimitot

Antimitot
ic
Non

1
1
1

n-Nitrosodimethylamine

6275-9598-

Methyl carbamate

55-0

Cyclophosphamide

6055-

monohydrate

19-2

Within-laboratory reproducibility

Non

Non

Non

1

Non

Non

Non

1

Non

Non

Non

1

100.0

100.0

100.0

(5/5)

(5/5)

(5/5)

(%)

Average100.0 (15/15)

Between-laboratory reproducibility (%)

92.0(Based on majority for Phase I)(23/25)9-7. The predictivity of the IL-2 Luc LTT in the Phase I and Phase II trials
When the predictivity of the IL-2 Luc LTT for immunosuppressive effects wasdetermined (Table 17), the IL-2 Luc LTT performance was 71.4 % (15/21) for meansensitivity, 100.0 % (4/4) for mean specificity, and 76.0 % (19/25) for mean prediction
for the combined data from the Phase I and Phase II trials (Table 18). On the other hand,when the predictivity of the IL-2 Luc LTT for antimitotic effects was determined (Table5), the IL-2 Luc LTT performance was 76.5 % (13/17) for mean sensitivity, 75.0 %(6/8) for mean specificity, and 76.0 % (19/25) for mean prediction for the combined
data from the Phase 1 and Phase 2 trials (Table 18).

72

Table 17. The judgment of each laboratory in the Phase I and Phase II trials

73

Table 18. The predictivity of each laboratory in the phase I and Phase II trials

7410. Discussion
10-1. Reliability
The IL-2 Luc LTT is based on the modulation of PMA/Io-induced luciferase
activity in the IL-2 reporter cell line, 2H4. Therefore, it is important that 2H4 cells
retain their ability to induce luciferase activity after PMA/Io stimulation after passage
for a sufficient number of times to perform the assay in the long term. We confirmedthat a frozen stock of 2H4 cells can be cultured without losing luciferase activity for atleast 16 weeks or 33 passages.
Culturing of 2H4 cells is relatively simple and does not require the use of trypsin
or EDTA because 2H4 cells do not adhere to the culture dishes. First, chemicals at
graded concentrations are added to the wells of a 96-well culture plate. Then, cells
adjusted to the optimum concentration are seeded into each well. After 24-hour
incubation, 100 μL of pre-warmed Tripluc Luciferase Assay Reagent is added to eachof the 96 wells. The subsequent process is completely automated, except for calculating
the results using the predesigned Excel spreadsheet. Therefore, the IL-2 Luc LTT is atest method that can significantly reduce human error.
Moreover, the IL-2 Luc LTT does not require the determination of cell viability
after chemical treatment. 2H4 cells can present IL-2 promoter activity as well as
promoter activity of GAPDH, a well-known housekeeping gene; therefore, information
regarding the effects of the chemical on both IL-2 promoter activity and cell viability is
obtained in each experiment. Furthermore, a single experiment takes only 31 hours,
including the time required for chemical preparation and cell plating.
75

10-2. Between- and within-laboratory reproducibility
We examined within-laboratory reproducibility in the Phase I trial. Lab A, Lab B,
and Lab C demonstrated 100%, 100%, and 100% reproducibility, respectively. On theother hand, the between-laboratory reproducibility result for Lab A, Lab B, and Lab C
was 92.0 % for the combined data of the Phase I and Phase II trials. These results
satisfied the acceptance criteria for the validation study of a within-laboratory
reproducibility of at least 80% and a between-laboratory reproducibility of at least 80%.

10-3. Predictivity
10-3-1. The predictivity of the IL-2 Luc LTT in the validation trials
In the combined data from the Phase 1 and Phase 2 trials, the IL-2 Luc LTT
performance for immunosuppressive effects was 71.4 % (15/21) for mean sensitivity,
100.0 % (4/4) for mean specificity, and 76.0 % (19/25) for mean prediction. On theother hand, the IL-2 Luc LTT performance for antimitotic effects was 76.5 % (13/17)
for mean sensitivity, 75.0 % (6/8) for mean specificity, and 76.0 % (19/25) for mean
prediction.

10-3-2. IL-2 Luc LTT judgment for 83 data set chemicals
To clarify the characteristics of the IL-2 Luc LTT, the lead laboratory assessed a
total of 85 test compounds including 46 pharmaceutical drugs that were composed of 12anti-cancer drugs, 8 immunosuppressive drugs with antimitotic effects, 876immunosuppressive drugs without antimitotic effects, 3 JAK inhibitors, and 15 nonimmunosuppressive drugs; and 37 non-pharmaceutical chemicals (23immunosuppressive and 14 non-immunosuppressive). Seven of the 23
immunosuppressive chemicals have antimitotic properties. (Kimura et al., 2021; Kimura
et al., 2020). To determine the performance of the IL-2 Luc LTT for the 37 nonpharmaceutical chemicals, we referred to reference data generated by collecting
literature information on the nine endpoints for each chemical (Appendix 14) andidentified chemicals that satisfy Criteria 1 (for immunosuppressive effects) or 2 (forantimitotic effects). The summarized immunotoxicity information, together with theclassification of each non-pharmaceutical chemicals, are shown in Appendix 14. The
judgment of 46 pharmaceutical drugs and 37 non-pharmaceutical chemicals by the IL-2
Luc LTT for antimitotic effects or for immunosuppressive effects is shown in Table 19
and Table 20, respectively. When pharmaceutical drugs were examined, theperformance of the IL-2 Luc LTT for antimitotic effects was 68.4 % (13/19) for
sensitivity, 91.7 % (22/24) for specificity, and 81.4 % (35/43) for predictivity (Table
24). When non-pharmaceutical chemicals were examined, the performance of the IL-2
Luc LTT for antimitotic effects was 50.0 % (4/8) for sensitivity, 88.9 % (24/27) for
specificity, and 80.0 % (28/35) for predictivity (Table 25). When pharmaceutical drugs
and non-pharmaceutical chemicals were examined together, the performance of the IL-2
Luc LTT for antimitotic effects was 63.0 % (17/27) for sensitivity, 90.2 % (46/51) for
specificity, and 80.8 % (63/78) for predictivity. When the applicability domain was
taken into consideration, the performance of the IL-2 Luc LTT for antimitotic effects
77was 62.5 % (15/24) for sensitivity, 90.2 % (46/51) for specificity, and 81.3 % (61/75)
for predictivity (Table 26).
On the other hand, when pharmaceutical drugs were examined, the performance of
the IL-2 Luc LTT for immunosuppressive effects was 43.3 % (13/30) for sensitivity,
84.6 % (11/13) for specificity, and 55.8 % (24/43) for predictivity (Table 25). When
non-pharmaceutical chemicals were examined, the performance of the IL-2 Luc LTT
for immunosuppressive effects was 26.1 % (6/23) for sensitivity, 91.7 % (11/12) for
specificity, and 48.6 % (17/35) for predictivity (Table 25). When pharmaceutical drugs
and non-pharmaceutical chemicals were examined together, the performance of the IL-2
Luc LTT for immunosuppressive effects was 35.8 % (19/53) for sensitivity, 88.0 %(22/25) for specificity, and 52.6 % (41/78) for predictivity. After considering theapplicability domain, the performance of the IL-2 Luc LTT for immunosuppressive
effects was 34.0 % (17/50) for sensitivity, 88.0 % (22/25) for specificity, and 52.0 %(39/75) for predictivity. (Table 26).

10-3-3. Application of the IL-2 Luc LTT with IL-2 Luc as part of an IATA
approach
To examine whether the combination of the IL-2 Luc LTT and the IL-2 Luc assay
improve the performance of the IL-2 Luc LTT for immunosuppressive effects, we firstexamined the performance of the IL-2 Luc assay alone (Table 21 and 23). The IL-2 Luc
assay that we have reported previously (Arch Toxicol 92: 2043-2054, 2018: Toxicol in
Vitro 66: 104832, 2020) and are currently under the validation is composed of three78

different endpoints: suppression, stimulation, and no effect. Since the IL-2 Luc assay is
to detect chemicals targeting T cell function, regardless of whether they are inhibitory
or stimulatory, stimulation was included as a potential judgement. In contrast, since theIL-2 Luc assay used in the combination with the IL-2 Luc LTT was aimed to detect
chemicals that induce immunosuppression, we considered stimulation judgment as noeffect. In Table 23, we presented the highest peak plasma concentrations (Cmax) of
each pharmaceutical drug and its lowest observed effect level (LOEL) values obtained
from the IL-2 Luc assay and the IL-2 Luc LTT. When pharmaceutical drugs were
examined, the performance of the IL-2 Luc assay for immunosuppressive effects was
48.4 % (15/31) for sensitivity, 66.7 % (10/15) for specificity, and 54.3 % (25/46) for
predictivity (Table 24). When non-pharmaceutical chemicals were examined, theperformance of the IL-2 Luc assay for immunosuppressive effects was 60.9 % (14/23)
for sensitivity, 64.3 % (9/14) for specificity, and 62.2 % (23/37) for predictivity (Table
25). When pharmaceutical drugs and non-pharmaceutical chemicals were examined
together, the performance of the IL-2 Luc assay for immunosuppressive effects was
53.7 % (29/54) for sensitivity, 59.0 % (19/29) for specificity, and 57.8 % (48/83) for
predictivity (Table 26).
Next, we combined these 2 assays based on the approach that drugs are judged as
immunosuppressive when at least one of these 2 assays gives positive results. Whenpharmaceutical drugs were examined, the performance of the combined assay for
immunosuppressive effects was 90.0 % (27/30) for sensitivity, 61.5 % (8/13) for
specificity, and 76.1% (33/43) for predictivity (Table 24). When non-pharmaceutical79

chemicals were examined, the performance of the combined assay for
immunosuppressive effects was 82.6 % (19/23) for sensitivity, 58.3 % (7/12) for
specificity, and 74.3 % (26/35) for predictivity (Table 25). When pharmaceutical drugs
and non-pharmaceutical chemicals were examined together, the performance of the
combined assay for immunosuppressive effects was 86.8 % (46/53) for sensitivity,
60.0 % (15/25) for specificity, and 78.2 % (61/78) for predictivity (Table 26). Afterconsidering the applicability domain, the performance of the combined assay was
88.8 % (44/50) for sensitivity, 60.0 % (15/25) for specificity, and 78.7 % (59/75) for
predictivity.

80

Table 19. IL-2 Luc LTT results and concordance with classification of 46
pharmaceutical drugs

81

Table 20. IL-2 Luc LTT results and concordance with literature-based classification for
non-pharmaceutical chemicals

82

Table 21. Combination of IL-2 Luc and IL-2 Luc LTT judgements and concordancewith classification of 46 pharmaceutical drugs.

83

Table 22. Comparison of in vivo Cmax and LOEL values from the IL-2 Luc and IL-2 Luc
LTT assays

84

Table 23. Combination of IL-2 Luc and IL-2 Luc LTT judgements and concordancewith literature-based classification for non-pharmaceutical chemicals

85

Table 24. The performance of the IL-2 Luc LTT, the IL-2 Luc assay, and the
combination when pharmaceutical drugs were examined

Table 25. The performance of the IL-2 Luc LTT, the IL-2 Luc assay, and the
combination when non-pharmaceutical chemicals were examined86Table 26. The performance of the IL-2 Luc LTT, the IL-2 Luc assay, and the
combination when pharmaceutical drugs and non-pharmaceutical chemicals were
examined

10-4. The possible reason for the false negative judgment for carmustin, paclitaxel,
leflunomide, and methotrexate (MTX) by the IL-2 Luc LTT.
The examination of 37 pharmaceutical drugs by the lead laboratory elucidated
three mechanisms for false negative judgments by the IL-2 Luc LTT. The first one isdue to significant suppression of the IL-2 Luc assay by drugs with antimitotic effects,
such as carmustin, paclitaxel, and leflunomide. Since the criteria of the IL-2 Luc LTT
for positive judgment includes “the mean of % suppression is ≤-35 (stimulatory) withstatistical significance”, the IL-2 Luc LTT cannot judge drugs that have inhibitory
effects on IL-2 transcription as positive which increase “the mean of % suppression is>35 with statistical significance”.
The second reason is due to the requirement of metabolic activation for drugs todemonstrate antimitotic effects. Of the 46 drugs tested, AZ, 6-mercaptoprine, andfludarabine are known to be metabolized to generate an active metabolite (Bradford and87Shih, 2011; Gandhi and Plunkett, 2002). The IL-2 Luc LTT judged AZ and 6meracaptoprine as non-antimitotic, while 6-thioguanine, the active metabolite of these 2
drugs, was judged as antimitotic. Although fludarabine was judged as positive by theIL-2 Luc LTT, the LOEL for Min Inh-GAPLA<0.7 was 250 µg/mL, which was muchhigher than the expected plasma concentration of the patients treated with fludarabine.
The third one is due to the mechanism underlying the false judgment of MTX.
MTX was also judged as negative because it did not show significant cytotoxicity evenat 200 µg/mL. Similar results were reported by Pessina et al. (Pessina et al., 2003) inwhich they could not determine the IC90 of MTX in either human umbilical cord blood
cells or murine bone marrow cells. There may be some underlying mechanisms in in
vitro culture that protect proliferating cells from MTX.

10-5. Limitation and applicability domain of the IL-2 Luc LTT
The IL-2 Luc LTT is applicable to test chemicals soluble or that form a stabledispersion but shares limitations common to many suspension cell-based assays when
testing highly hydrophobic substances. Chemicals that interfere with luciferase or
luminescence can confound its activity/measurement (Thorne et al. 2010) and thus thesechemicals are out of the applicability domain. In addition, the following limitations
should be noted: 1) the use of PMA/Io as a stimulant bypasses signaling through the Tcell receptor and the subsequent intracellular signaling events that precede activation ofphospholipase C, and therefore precludes detection of chemicals that act on those88

upstream signaling molecules (Ohtsuka et al., 1996); (2) the Jurkat T cell line (fromwhich 2H4 cells are derived) are demonstrated to be suitable for examining themolecular mechanism underlying immunotoxicity (Shao et al., 2013), they may lackseveral key proteins involved in the activation of normal T cells in response to TCR
stimulation, and therefore may not be able to detect effects of chemicals that act on
those key proteins.
Of the 46 drugs tested, AZ, 6-mercaptoprine, and fludarabine are known to be
metabolized to generate the active metabolite (Bradford and Shih, 2011; Gandhi andPlunkett, 2002). The IL-2 Luc LTT judged AZ and 6-meracaptoprine as nonantimitotic, while 6-thioguanine, the active metabolite of these 2 drugs, was judged as
antimitotic. Although fludarabine was judged as positive by the IL-2 Luc LTT, thelowest observed effect level (LOEL) for Min Inh-GAPLA<0.7 was 250 µg/mL, which
was much higher than the expected plasma concentration of the patients treated withfludarabine. 2H4 cells are not supposed to express the enzymes required to metabolize
and activate drugs and thus drugs that need metabolic activation are out of theapplicability domain.

10-6. The possible reason for the poor specificity of the IL-2 Luc LTT
Although the sensitivity and the predictivity of the combination assay seemed to be
acceptable, the specificity was not satisfactory. In general, there is a marked differencein the concentrations of chemicals used in in vivo and in vitro tests. Usually, muchhigher concentrations can be used in vitro compared with in vivo. Indeed, the IL-2 Luc
89

LTT and IL-2 Luc assay examined the effects of chemicals starting at the highest
concentration of 200 μg/mL. In contrast, the highest peak plasma concentrations(Cmax) of the 46 pharmaceutical drugs using customary dose regimes were <50 μg/mL.
In addition, it is unlikely that the plasma concentration of non-pharmaceutical chemicals
will exceed that of pharmaceutical drugs in most cases. Therefore, it is likely that falsepositive judgments by the IL-2 Luc assay or IL-2 Luc LTT are due to the effects ofchemicals at concentrations that have not been examined in in vivo experiments, thus
resulting in low assay specificity (Fig. 8).

Fig. 8. The possible reason for the poor specificity of the IL-2 Luc LTT

As long as exact LOELs cannot be obtained, the IL-2 Luc assay, the IL-2 Luc LTT,
and their combination can be used only for hazard identification. However, although theexact LOELs were not known, the dissociation between the LOELs and Cmax values ofanti-cancer drugs or immunosuppressive drugs tends to be much smaller than that of90

non-immunosuppressive drugs. We therefore calculated the ratio of LOEL/Cmax of thedrugs and found that 22 of the 26 (84.6%) anti-cancer and immunosuppressive drugs
and only 1 of the 7 (14.3%) non-immunosuppressive drugs showed a LOEL/Cmax ratio
below 15 (Fig. 9). If we tentatively judged the drugs whose LOELs were more than 15
times higher than Cmax as negative, the performance of the combined assay was 88.9 %(24/27) for sensitivity, 92.3 % (12/13) for specificity and 90.0 % (36/40) for accuracy
after considering the applicability domain.

Fig. 9. The ratio of LOEL/Cmax for each drug.

91

10-7. Regulatory application of the IL-2 Luc LTT
The CAS REGISTRYSM currently contains more than 130 million unique organicand inorganic chemical substances, such as alloys, coordination compounds, minerals,
mixtures, polymers, and salts. Humans are exposed to many of these substances, which
are present as environmental contaminants or used as food additives and drugs. Some ofthese compounds can target the immune system, resulting in adverse health effects suchas the development of allergies, autoimmune disorders, increased susceptibility toinfection and cancer due to immunosuppression. Accordingly, immunotoxicity, which isdefined as the toxicological effects of xenobiotics on the function of the immune
system, is a matter of serious concern to the public as well as regulatory agencies. To
address these concerns, the World Health Organization published its Guidance for
Immunotoxicity Risk Assessment for Chemicals (World Health Organization (WHO,2012). Currently, the assessment of chemical immunotoxicity relies mainly on animalmodels and assays that characterize immunosuppression and sensitization. However,animal trials have many drawbacks, such as high cost, ethical concerns, andquestionable relevance to risk assessment for humans, that they cannot be used to screen
the immunotoxicity of more than 130 million chemicals. Therefore, there is an urgent
need to develop alternative testing methods and assessment strategies to reduce the useof laboratory animals and, if possible, replace animals used in scientific trials (Adler et
al. 2011). To date, however, there are no OECD test guidelines to detect chemical
immunotoxicity in vitro. We would therefore like to propose the IL-2 Luc LTT in

92

addition to the IL-2 Luc assay an in vitro alternative test methods for immunotoxicity,
especially immunosuppression by chemicals.

11. Conclusion
We conducted the validation of the IL-2 Luc LTT and could demonstrate theacceptable within- and between-laboratory reproducibility of this assay. The IL-2 Luc
LTT can detect immunosuppressive effects of chemicals caused by the antimitotic effectthat cannot be detected by the IL-2 Luc assay. Indeed, the combination of these two
assays increases the identification of most immunosuppressive drugs used for organtransplantation, bone marrow transplantation, or treatment of collagen diseases. The
performance of the combination of the two assays together for pharmaceutical drugs
improves to 95.0% (38/40) after considering the applicability domain. Even for the
immunosuppressive effects of non-pharmaceutical chemicals, the performance of the
combination of assays is improved to 74.3 % (26/35).
Considering that compounds can target different pathways and immune cells,
the assessment of immunotoxicity will require the combination with other assays tofurther improve the predictivity. The IL-2 Luc LTT is an indispensable component for
the IATA for immunotoxicity. We would therefore like to propose the IL-2 Luc LTT as
the OECD test guideline for in vitro immunotoxicity tests.

9312. Acknowledgement
This validation study was supported by Grants-in-Aid from the Ministry of
Economy, Trade and Industry (METI), the Ministry of Health, Labor and Welfare(MHLW), and the Japanese Society for Alternatives to Animal Experiments (JSAAE).
We gratefully acknowledge voluntary work by the participating laboratories and theEuropean Union Reference Laboratory for Alternatives to Animal Testing (EURLECVAM), and the NTP Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM).

94

13. References
Adler, S., Basketter, D., Creton, S., et al. (2011), Alternative (non-animal) methods forcosmetics testing: current status and future prospects-2010. Arch toxicol 85: 367485, 10.1007/s00204-011-0693-2
Allison, A.C. (2000), Immunosuppressive drugs: the first 50 years and a glance forward.
Immunopharmacol 47: 63-83,
Barber, R.D., Harmer, D.W., Coleman, R.A., et al. (2005), GAPDH as a housekeeping
gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues.
Physiol Genomics 21: 389-395, 10.1152/physiolgenomics.00025.2005
Bradford, K., Shih, D.Q. (2011), Optimizing 6-mercaptopurine and azathioprine therapy
in the management of inflammatory bowel disease. World J Gastroenterol 17:
4166-4173, 10.3748/wjg.v17.i37.4166
Edwards, D.R., Denhardt, D.T. (1985), A study of mitochondrial and nuclear
transcription with cloned cDNA probes. Changes in the relative abundance of
mitochondrial transcripts after stimulation of quiescent mouse fibroblasts. Exp Cell
Res 157: 127-143,
Galbiati, V., Mitjans, M., Corsini, E. (2010), Present and future of in vitro
immunotoxicology in drug development. J Immunotoxicol 7: 255-267,
10.3109/1547691X.2010.509848
Gandhi, V., Plunkett, W. (2002), Cellular and clinical pharmacology of fludarabine. Clin
Pharmacokinet 41: 93-103, 10.2165/00003088-200241020-00002
Gennari, A., Ban, M., Braun, A., et al. (2005), The Use of In Vitro Systems for
Evaluating Immunotoxicity: The Report and Recommendations of an ECVAM
Workshop. J Immunotoxicoll 2: 61-83, 10.1080/15476910590965832
Kimura, Y., Fujimura, C., Ito, Y., et al. (2014), Evaluation of the Multi-ImmunoTox
Assay composed of 3 human cytokine reporter cells by examining immunologicaleffects of drugs.Toxicol In Vitro 28: 759-768, 10.1016/j.tiv.2014.02.013
Kimura, Y., Fujimura, C., Ito, Y., et al. (2015), Optimization of the IL-8 Luc assay as anin vitro test for skin sensitization. Antimitoticol In Vitro 29: 1816-1830,
10.1016/j.tiv.2015.07.006
Kimura, Y., Fujimura, C., Ito, Y., et al. (2018), Profiling the immunotoxicity ofchemicals based on in vitro evaluation by a combination of the Multi-ImmunoTox

95

assay and the IL-8 Luc assay. Arch Toxicl 92: 2043-2054, 10.1007/s00204-0182199-7
Kimura, Y., Terui, H., Fujimura, C., et al. (2021), Optimization of the IL-2 Luc assay for
immunosuppressive drugs: a novel in vitro immunotoxicity test with highsensitivity and predictivity. Arch Toxicol 95: 2755-2768, 10.1007/s00204-02103101-4
Kimura, Y., Yasuno, R., Watanabe, M., et al. (2020), An international validation study ofthe IL-2 Luc assay for evaluating the potential immunotoxic effects of chemicals on
T cells and a proposal for reference data for immunotoxic chemicals. Antimitoticol
In Vitro 66: 104832, 10.1016/j.tiv.2020.104832
Langezaal, I., Hoffmann, S., Hartung, T., et al. (2002), Evaluation and prevalidation of
an immunotoxicity test based on human whole-blood cytokine release. Alternatives
to laboratory animals : ATLA 30: 581-595,
Lankveld, D.P., Van Loveren, H., Baken, K.A., et al. (2010), In vitro testing for direct
immunotoxicity: state of the art. Meth Mol Biol 598: 401-423, 10.1007/978-160761-401-2_26
Luster, A.D. (1998), Chemokines--chemotactic cytokines that mediate inflammation.
New Eng J Med 338: 436-445,
Luster, M.I., Portier, C., Pait, D.G., et al. (1993), Risk assessment in immunotoxicology.
II. Relationships between immune and host resistance tests. Fundam Appl Toxicol
21: 71-82
Portier, C., Pait, D.G., et al. (1992), Risk assessment in immunotoxicology. I.
Sensitivity and predictability of immune tests. Fundam Appl Toxicol 18: 200-210,10.1016/0272-0590(92)90047-l
Michelini, E., Cevenini, L., Calabretta, M.M., et al. (2014), Exploiting in vitro and invivo bioluminescence for the implementation of the three Rs principle(replacement, reduction, and refinement) in drug discovery. Anal Bioanal Chem
406: 5531-5539, 10.1007/s00216-014-7925-2
Mori, R., Wang, Q., Danenberg, K.D., et al. (2008), Both beta-actin and GAPDH are
useful reference genes for normalization of quantitative RT-PCR in human FFPE
tissue samples of prostate cancer. Prostate 68: 1555-1560, 10.1002/pros.20815
Nakajima, Y., Ohmiya, Y. (2010), Bioluminescence assays: multicolor luciferase assay,
secreted luciferase assay and imaging luciferase assay. Expert Opin Drug Discov 5:96835-849, 10.1517/17460441.2010.506213
OECD 2018. Good Cell Culture Practice (GCCP)", in Guidance Document on Good In
Vitro Method Practices (GIVIMP), OECD Guidance document on Good In vitro
Method Practices (GIVIMP). OECD publishing. Paris.
Oliveira, J.G., Prados, R.Z., Guedes, A.C., et al. (1999), The housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase is inappropriate as internal control incomparative studies between skin tissue and cultured skin fibroblasts using
Northern blot analysis. Arch Dermatol Res 291: 659-661,
Pessina, A., Albella, B., Bayo, M., et al. (2003), Application of the CFU-GM assay to
predict acute drug-induced neutropenia: an international blind trial to validate aprediction model for the maximum tolerated dose (MTD) of myelosuppressive
xenobiotics. Toxicol Sci 75: 355-367, 10.1093/toxsci/kfg188
Roda, A., Pasini, P., Mirasoli, M., et al. (2004), Biotechnological applications of
bioluminescence and chemiluminescence. Trends Biotechnol 22: 295-303,
10.1016/j.tibtech.2004.03.011
Saito, R., Hirakawa, S., Ohara, H., et al. (2011), Nickel differentially regulates NFAT
and NF-kappaB activation in T cell signaling. Toxicol Appl Pharamacol 254: 245255, 10.1016/j.taap.2011.04.017
Shao, J., Berger, L.F., Hendriksen, P.J., et al. (2014), Transcriptome-based functionalclassifiers for direct immunotoxicity. Arch Toxicol 88: 673-689, 10.1007/s00204013-1179-1
Takahashi, T., Kimura, Y., Saito, R., et al. (2011), An in vitro test to screen skinsensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8. Toxicol
Sci 124: 359-369, 10.1093/toxsci/kfr237
Thellin, O., Zorzi, W., Lakaye, B., et al. (1999), Housekeeping genes as internal
standards: use and limits. J Biotechnol 75: 291-295,
Wagner, W., Walczak-Drzewiecka, A., Slusarczyk, A., et al. (2006), Fluorescent Cell
Chip a new in vitro approach for immunotoxicity screening. Toxicol Lett 162: 5570, 10.1016/j.toxlet.2005.10.017
WHO (2012), Guidance for immunotoxicity risk assessment for chemicals. WHO press,
Paris,
Winer, J., Jung, C.K., Shackel, I., et al. (1999), Development and validation of real-time97

quantitative reverse transcriptase-polymerase chain reaction for monitoring gene
expression in cardiac myocytes in vitro. Anal Biochem 270: 41-49,
10.1006/abio.1999.4085

14. List of abbreviations
95% CI : the 95% confidence interval
AIST : National Institute of Advanced Industrial Science and Technology
AOP : Adverse outcome pathway
ARE: Antioxidant response element
CAS No. : Chemical Abstract Service Number
CMV : Cytomegalovirus
CSC : the Chemical Selection Committee
DMSO : Dimethyl sulfoxide
DPRA : the Direct Peptide Reactivity Assay
ECVAM : the European Centre for Validation of Alternative Methods
EDTA : Ethylenediaminetetraacetic acid
EGFR : Epidermal growth factor receptor
EGR-1 : Early growth response-1
EU : European Union
FBS : Fetal bovine serum
FN : False Negative Rate
GLP : Good laboratory Practice
GSH : Glutathione
HRI/FDSC : Hatano Research Institute, Food & Drug Safety Center
HSV : Herpes simplex viruses
ICCVAM : Interagency Coordinating Committee on the Validation of Alternative
Methods
ID : Identification
IFN-γ: Interferon-γ
I.I.-SLR-LA : Inhibition index of SLR-LA
IL-2 : Interleukin-2
IL-8 : Interleukin-8
98

JaCVAM : the Japanese Center for the Validation of Alternative Methods
JSIT : Japanese Society of Immunotoxicology
Keap-1 : Kelch-like ECH-associated protein 1
KoCVAM : Korean Center for the Validation of Alternative Methods
LLNA : Local lymph node assay
LPS : Lipopolysaccharide
MIT : Minimum induction threshold
MITA : Multi-ImmunoTox Assay
MoDCs : Monocyte-derived dendritic cells
MOVS: Management Office of Validation StudymRNA : messenger ribonucleic acid
MSDS : Material safety data sheet
NICEATM : the National Toxicology Program Interagency Center for the Evaluation of
Alternative Toxicological Methods
NIHS : National Institute of Health Sciences
NPV : Negative predictive value
Nqo1 : NADPH-quinone oxidoreductase 1
Nrf2 : Nuclear factor (erythroid-derived 2)-like factor 2
nSLG-LA : normalized SLG luciferase activity
nSLO-LA : normalized SLO luciferase activity
OECD : the Organization for Economic Co-operation and Development
PCR : Polymerase chain reaction
PI : Propidium iodide
PMA/Io : Phorbol 12-myristate 13-acetate/Ionomycin
PN : False Positive Rate
PPV : Positive Predictive Value
QC : Quality Control
REACH : Registration, Evaluation, Authorization and Restriction of Chemicals
RFI : Relative fluorescence intensity
RT : Ring trial
SLG : Stable luciferase green
SLG-LA : SLG luciferase activity
SLO : Stable luciferase orange
99

SLO-LA : SLO luciferase activity
SLR : Stable luciferase red
SLR-LA : SLR luciferase activity
SLS : Sodium lauryl sulfate
SLR : Stable luciferase red
SLR-LA : SLR luciferase activity
SV40 : Simian virus 40
TG : Test Guideline
TNF-α : Tumor necrosis factor-α
UN GHS : the United Nations Globally Harmonized System of Classification andLabeling of Chemicals
VMT : Validation Management Team100